DNA mutagenesis by random fragmentation and reassembly

ABSTRACT

A method for generating libraries of displayed peptides and/or antibodies (Abs) suitable for affinity interaction screening or phenotypic screening comprising: (i) obtaining selected library members comprising a displayed peptide and/or Ab and the corressponding polynucleotide (PN), or copies of it, (ii) pooling and fragmenting the PN, or copies of it, to form fragments, (iii) performing PCR amplification and thereby homologously recombining the fragments to form a shuffled pool of recombined PNs, which are not present in the selected library of (i).

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a method for the production ofpolynucleotides conferring a desired phenotype and/or encoding a proteinhaving an advantageous predetermined property which is selectable. In anaspect, the method is used for generating and selecting nucleic acidfragments encoding mutant proteins.

2. Description of the Related Art

The complexity of an active sequence of a biological macromolecule, e.g.proteins, DNA etc., has been called its information content ("IC"; 5-9).The information content of a protein has been defined as the resistanceof the active protein to amino acid sequence variation, calculated fromthe minimum number of invariable amino acids (bits) required to describea family of related sequences with the same function (9, 10). Proteinsthat are sensitive to random mutagenesis have a high informationcontent. In 1974, when this definition was coined, protein diversityexisted only as taxonomic diversity.

Molecular biology developments such as molecular libraries have allowedthe identification of a much larger number of variable bases, and evento select functional sequences from random libraries. Most residues canbe varied, although typically not all at the same time, depending oncompensating changes in the context. Thus a 100 amino acid protein cancontain only 2,000 different mutations, but 20¹⁰⁰ possible combinationsof mutations.

Information density is the Information Content/unit length of asequence. Active sites of enzymes tend to have a high informationdensity. By contrast, flexible linkers in enzymes have a low informationdensity (8).

Current methods in widespread use for creating mutant proteins in alibrary format are error-prone polymerase chain reaction (11, 12, 19)and cassette mutagenesis (8, 20, 21, 22, 40, 41, 42), in which thespecific region to be optimized is replaced with a syntheticallymutagenized oligonucleotide. In both cases, a `mutant cloud` (4) isgenerated around certain sites in the original sequence.

Error-prone PCR uses low-fidelity polymerization conditions to introducea low level of point mutations randomly over a long sequence. Errorprone PCR can be used to mutagenize a mixture of fragments of unknownsequence. However, computer simulations have suggested that pointmutagenesis alone may often be too gradual to allow the block changesthat are required for continued sequence evolution. The publishederror-prone PCR protocols do not allow amplification of DNA fragmentsgreater than 0.5 to 1.0 kb, limiting their practical application.Further, repeated cycles of error-prone PCR lead to an accumulation ofneutral mutations, which, for example, may make a protein immunogenic.

In oligonucleotide-directed mutagenesis, a short sequence is replacedwith a synthetically mutagenized oligonucleotide. This approach does notgenerate combinations of distant mutations and is thus notcombinatorial. The limited library size relative to the vast sequencelength means that many rounds of selection are unavoidable for proteinoptimization. Mutagenesis with synthetic oligonucleotides requiressequencing of individual clones after each selection round followed bygrouping into families, arbitrarily choosing a single family, andreducing it to a consensus motif, which is resynthesized and reinsertedinto a single gene followed by additional selection. This processconstitutes a statistical bottleneck, it is labor intensive and notpractical for many rounds of mutagenesis.

Error-prone PCR and oligonucleotide-directed mutagenesis are thus usefulfor single cycles of sequence fine tuning but rapidly become limitingwhen applied for multiple cycles.

Error-prone PCR can be used to mutagenize a mixture of fragments ofunknown sequence (11, 12). However, the published error-prone PCRprotocols (11, 12) suffer from a low processivity of the polymerase.Therefore, the protocol is unable to result in the random mutagenesis ofan average-sized gene. This inability limits the practical applicationof error-prone PCR.

Another serious limitation of error-prone PCR is that the rate ofdown-mutations grows with the information content of the sequence. At acertain information content, library size, and mutagenesis rate, thebalance of down-mutations to up-mutations will statistically prevent theselection of further improvements (statistical ceiling).

Finally, repeated cycles of error-prone PCR will also lead to theaccumulation of neutral mutations, which can affect, for example,immunogenicity but not binding affinity.

Thus error-prone PCR was found to be too gradual to allow the blockchanges that are required for continued sequence evolution (1, 2).

In cassette mutagenesis, a sequence block of a single template istypically replaced by a (partially) randomized sequence. Therefore, themaximum information content that can be obtained is statisticallylimited by the number of random sequences (i.e., library size). Thisconstitutes a statistical bottleneck, eliminating other sequencefamilies which are not currently best, but which may have greater longterm potential.

Further, mutagenesis with synthetic oligonucleotides requires sequencingof individual clones after each selection round (20). Therefore, thisapproach is tedious and is not practical for many rounds of mutagenesis.

Error-prone PCR and cassette mutagenesis are thus best suited and havebeen widely used for fine-tuning areas of comparatively low informationcontent. One apparent exception is the selection of an RNA ligaseribozyme from a random library using many rounds of amplification byerror-prone PCR and selection (13).

It is becoming increasingly clear that the tools for the design ofrecombinant linear biological sequences such as protein, RNA and DNA arenot as powerful as the tools nature has developed. Finding better andbetter mutants depends on searching more and more sequences withinlarger and larger libraries, and increasing numbers of cycles ofmutagenic amplification and selection are necessary. However asdiscussed above, the existing mutagenesis methods that are in widespreaduse have distinct limitations when used for repeated cycles.

Evolution of most organisms occurs by natural selection and sexualreproduction. Sexual reproduction ensures mixing and combining of thegenes of the offspring of the selected individuals. During meiosis,homologous chromosomes from the parents line up with one another andcross-over part way along their length, thus swapping genetic material.Such swapping or shuffling of the DNA allows organisms to evolve morerapidly (1, 2). In sexual recombination, because the inserted sequenceswere of proven utility in a homologous environment, the insertedsequences are likely to still have substantial information content oncethey are inserted into the new sequence.

Marton et al., (27) describes the use of PCR in vitro to monitorrecombination in a plasmid having directly repeated sequences. Marton etal. discloses that recombination will occur during PCR as a result ofbreaking or nicking of the DNA. This will give rise to recombinantmolecules. Meyerhans et al. (23) also disclose the existence of DNArecombination during in vitro PCR.

The term Applied Molecular Evolution ("AME") means the application of anevolutionary design algorithm to a specific, useful goal. While manydifferent library formats for AME have been reported for polynucleotides(3, 11-14), peptides and proteins (phage (15-17), lacI (18) andpolysomes, in none of these formats has recombination by randomcross-overs been used to deliberately create a combinatorial library.

Theoretically there are 2,000 different single mutants of a 100 aminoacid protein. A protein of 100 amino acids has 20¹⁰⁰ possiblecombinations of mutations, a number which is too large to exhaustivelyexplore by conventional methods. It would be advantageous to develop asystem which would allow the generation and screening of all of thesepossible combination mutations.

Winter and coworkers (43,44) have utilized an in vivo site specificrecombination system to combine light chain antibody genes with heavychain antibody genes for expression in a phage system. However, theirsystem relies on specific sites of recombination and thus is limited.Hayashi et al. (48) report simultaneous mutagenesis of antibody CDRregions in single chain antibodies (scFv) by overlap extension and PCR.

Caren et al. (45) describe a method for generating a large population ofmultiple mutants using random in vivo recombination. However, theirmethod requires the recombination of two different libraries ofplasmids, each library having a different selectable marker. Thus themethod is limited to a finite number of recombinations equal to thenumber of selectable markers existing, and produces a concomitant linearincrease in the number of marker genes linked to the selectedsequence(s).

Calogero et al. (46) and Galizzi et al. (47) report that in vivorecombination between two homologous but truncated insect-toxin genes ona plasmid can produce a hybrid gene. Radman et al. (49) report in vivorecombination of substantially mismatched DNA sequences in a host cellhaving defective mismatch repair enzymes, resulting in hybrid moleculeformation.

It would be advantageous to develop a method for the production ofmutant proteins which method allowed for the development of largelibraries of mutant nucleic acid sequences which were easily searched.The invention described herein is directed to the use of repeated cyclesof point mutagenesis, nucleic acid shuffling and selection which allowfor the directed molecular evolution in vitro of highly complex linearsequences, such as proteins through random recombination.

Accordingly, it would be advantageous to develop a method which allowsfor the production of large libraries of mutant DNA, RNA or proteins andthe selection of particular mutants for a desired goal. The inventiondescribed herein is directed to the use of repeated cycles ofmutagenesis, in vivo recombination and selection which allow for thedirected molecular evolution in vivo of highly complex linear sequences,such as DNA, RNA or proteins through recombination.

Further advantages of the present invention will become apparent fromthe following description of the invention with reference to theattached drawings.

SUMMARY OF THE INVENTION

The present invention is directed to a method for generating a selectedpolynucleotide sequence or population of selected polynucleotidesequences, typically in the form of amplified and/or clonedpolynucleotides, whereby the selected polynucleotide sequence(s) possessa desired phenotypic characteristic (e.g., encode a polypeptide, promotetranscription of linked polynucleotides, bind a protein, and the like)which can be selected for. One method of identifying polypeptides thatpossess a desired structure or functional property, such as binding to apredetermined biological macromolecule (e.g., a receptor), involves thescreening of a large library of polypeptides for individual librarymembers which possess the desired structure or functional propertyconferred by the amino acid sequence of the polypeptide.

The present invention provides a method for generating libraries ofdisplayed polypeptides or displayed antibodies suitable for affinityinteraction screening or phenotypic screening. The method comprises (1)obtaining a first plurality of selected library members comprising adisplayed polypeptide or displayed antibody and an associatedpolynucleotide encoding said displayed polypeptide or displayedantibody, and obtaining said associated polynucleotides or copiesthereof wherein said associated polynucleotides comprise a region ofsubstantially identical sequence, optionally introducing mutations intosaid polynucleotides or copies, and (2) pooling and fragmenting,typically randomly, said associated polynucleotides or copies to formfragments thereof under conditions suitable for PCR amplification,performing PCR amplification and optionally mutagenesis, and therebyhomologously recombining said fragments to form a shuffled pool ofrecombined polynucleotides, whereby a substantial fraction (e.g.,greater than 10 percent) of the recombined polynucleotides of saidshuffled pool are not present in the first plurality of selected librarymembers, said shuffled pool composing a library of displayedpolypeptides or displayed antibodies suitable for affinity interactionscreening. Optionally, the method comprises the additional step ofscreening the library members of the shuffled pool to identifyindividual shuffled library members having the ability to bind orotherwise interact (e.g., such as catalytic antibodies) with apredetermined macromolecule, such as for example a proteinaceousreceptor, peptide, oligosaccharide, virion, or other predeterminedcompound or structure. The displayed polypeptides, antibodies,peptidomimetic antibodies, and variable region sequences that areidentified from such libraries can be used for therapeutic, diagnostic,research, and related purposes (e.g., catalysts, solutes for increasingosmolarity of an aqueous solution, and the like), and/or can besubjected to one or more additional cycles of shuffling and/or affinityselection. The method can be modified such that the step of selecting isfor a phenotypic characteristic other than binding affinity for apredetermined molecule (e.g., for catalytic activity, stability,oxidation resistance, drug resistance, or detectable phenotype conferredon a host cell).

In one embodiment, the first plurality of selected library members isfragmented and homologously recombined by PCR in vitro.

In one embodiment, the first plurality of selected library members isfragmented in vitro, the resultant fragments transferred into a hostcell or organism and homologously recombined to form shuffled librarymembers in vivo.

In one embodiment, the first plurality of selected library members iscloned or amplified on episomally replicable vectors, a multiplicity ofsaid vectors is transferred into a cell and homologously recombined toform shuffled library members in vivo.

In one embodiment, the first plurality of selected library members isnot fragmented, but is cloned or amplified on an episomally replicablevector as a direct repeat, which each repeat comprising a distinctspecies of selected library member sequence, said vector is transferredinto a cell and homologously recombined by intra-vector recombination toform shuffled library members in vivo.

In an embodiment, combinations of in vitro and in vivo shuffling areprovided to enhance combinatorial diversity.

The present invention provides a method for generating libraries ofdisplayed antibodies suitable for affinity interaction screening. Themethod comprises (1) obtaining a first plurality of selected librarymembers comprising a displayed antibody and an associated polynucleotideencoding said displayed antibody, and obtaining said associatedpolynucleotides or copies thereof, wherein said associatedpolynucleotides comprise a region of substantially identical variableregion framework sequence, and (2) pooling and fragmenting saidassociated polynucleotides or copies to form fragments thereof underconditions suitable for PCR amplification and thereby homologouslyrecombining said fragments to form a shuffled pool of recombinedpolynucleotides comprising novel combinations of CDRs, whereby asubstantial fraction (e.g., greater than 10 percent) of the recombinedpolynucleotides of said shuffled pool comprise CDR combinations are notpresent in the first plurality of selected library members, saidshuffled pool composing a library of displayed antibodies comprising CDRpermutations and suitable for affinity interaction screening.Optionally, the shuffled pool is subjected to affinity screening toselect shuffled library members which bind to a predetermined epitope(antigen) and thereby selecting a plurality of selected shuffled librarymembers. Optionally, the plurality of selected shuffled library memberscan be shuffled and screened iteratively, from 1 to about 1000 cycles oras desired until library members having a desired binding affinity areobtained.

Accordingly, one aspect of the present invention provides a method forintroducing one or more mutations into a template double-strandedpolynucleotide, wherein the template double-stranded polynucleotide hasbeen cleaved into random fragments of a desired size, by adding to theresultant population of double-stranded fragments one or more single ordouble-stranded oligonucleotides, wherein said oligonucleotides comprisean area of identity and an area of heterology to the templatepolynucleotide; denaturing the resultant mixture of double-strandedrandom fragments and oligonucleotides into single-stranded fragments;incubating the resultant population of single-stranded fragments with apolymerase under conditions which result in the annealing of saidsingle-stranded fragments at regions of identity between thesingle-stranded fragments and formation of a mutagenized double-strandedpolynucleotide; and repeating the above steps as desired. In anotheraspect the present invention is directed to a method of producingrecombinant proteins having biological activity by treating a samplecomprising double-stranded template polynucleotides encoding a wild-typeprotein under conditions which provide for the cleavage of said templatepolynucleotides into random double-stranded fragments having a desiredsize; adding to the resultant population of random fragments one or moresingle or double-stranded oligonucleotides, wherein saidoligonucleotides comprise areas of identity and areas of heterology tothe template polynucleotide; denaturing the resultant mixture ofdouble-stranded fragments and oligonucleotides into single-strandedfragments; incubating the resultant population of single-strandedfragments with a polymerase under conditions which result in theannealing of said single-stranded fragments at the areas of identity andformation of a mutagenized double-stranded polynucleotide; repeating theabove steps as desired; and then expressing the recombinant protein fromthe mutagenized double-stranded polynucleotide.

A third aspect of the present invention is directed to a method forobtaining a chimeric polynucleotide by treating a sample comprisingdifferent double-stranded template polynucleotides wherein saiddifferent template polynucleotides contain areas of identity and areasof heterology under conditions which provide for the cleavage of saidtemplate polynucleotides into random double-stranded fragments of adesired size; denaturing the resultant random double-stranded fragmentscontained in the treated sample into single-stranded fragments;incubating the resultant single-stranded fragments with polymerase underconditions which provide for the annealing of the single-strandedfragments at the areas of identity and the formation of a chimericdouble-stranded polynucleotide sequence comprising templatepolynucleotide sequences; and repeating the above steps as desired.

A fourth aspect of the present invention is directed to a method ofreplicating a template polynucleotide by combining in vitrosingle-stranded template polynucleotides with small randomsingle-stranded fragments resulting from the cleavage and denaturationof the template polynucleotide, and incubating said mixture of nucleicacid fragments in the presence of a nucleic acid polymerase underconditions wherein a population of double-stranded templatepolynucleotides is formed.

The invention also provides the use of polynucleotide shuffling, invitro and/or in vivo to shuffle polynculeotides encoding polypeptidesand/or polynucleotides comprising transcriptional regulatory sequences.

The invention also provides the use of polynucleotide shuffling toshuffle a population of viral genes (e.g., capsid proteins, spikeglycoproteins, polymerases, proteases, etc.) or viral genomes (e.g.,paramyxoviridae, orthomyxoviridae, herpesviruses, retroviruses,reoviruses, rhinoviruses, etc.). In an embodiment, the invnetionprovides a method for shuffling sequences encoding all or portions ofimmunogenic viral proteins to generate novel combinations of epitopes aswell as novel epitopes created by recombination; such shuffled viralproteins may comprise epitopes or combinations of epitopes which arelikely to arise in the natural environment as a consequence of viralevolution (e.g., such as recombination of influenza virus strains).

The invention also provides a method suitable for shufflingpolynucleotide sequences for generating gene therapy vectors andreplication-defective gene therapy constructs, such as may be used forhuman gene therapy, including but not limited to vaccination vectors forDNA-based vaccination, as well as anti-neoplastic gene therapy and othergene therapy formats.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram comparing mutagenic shuffling overerror-prone PCR; (a) the initial library; (b) pool of selected sequencesin first round of affinity selection; (d) in vitro recombination of theselected sequences (`shuffling`); (f) pool of selected sequences insecond round of affinity selection after shuffling; (c) error-prone PCR;(e) pool of selected sequences in second round of affinity selectionafter error-prone PCR.

FIG. 2 illustrates the reassembly of a 1.0 kb LacZ alpha gene fragmentfrom 10-50 bp random fragments. (a) Photograph of a gel of PCR amplifiedDNA fragment having the LacZ alpha gene. (b) Photograph of a gel of DNAfragments after digestion with DNAseI. (c) Photograph of a gel of DNAfragments of 10-50 bp purified from the digested LacZ alpha gene DNAfragment; (d) Photograph of a gel of the 10-50 bp DNA fragments afterthe indicated number of cycles of DNA reassembly; (e) Photograph of agel of the recombination mixture after amplification by PCR withprimers.

FIGS. 3a and 3b are schematic illustrations of the LacZ alpha gene stopcodon mutants and their DNA sequences. The boxed regions areheterologous areas, serving as markers. The stop codons are located insmaller boxes or underlined. "+" indicates a wild-type gene and "-"indicates a mutated area in the gene.

FIG. 4 is a schematic illustration of the introduction or spiking of asynthetic oligonucleotide into the reassembly process of the LacZ alphagene.

FIGS. 5a and 5b illustrate the regions of homology between a murineIL1-B gene (M) and a human IL1-B gene (H) with E. coli codon usage.Regions of heterology are boxed. The "₋₋ |⁻⁻ " indicate crossoversobtained upon the shuffling of the two genes.

FIG. 6 is a schematic diagram of the antibody CDR shuffling model systemusing the scFv of anti-rabbit IgG antibody (A10B).

FIG. 7 illustrates the observed frequency of occurrence of certaincombinations of CDRs in the shuffled DNA of the scFv of anti-rabbit IgGantibody (A10B).

FIG. 8 illustrates the improved avidity of the scFv anti-rabbit antibodyafter DNA shuffling and each cycle of selection.

FIG. 9 schematically portrays pBR322-Sfi-BL-LA-Sfi and in vivointraplasmidic recombination via direct repeats, as well as the rate ofgeneration of ampicillin-resistant colonies by intraplasmidicrecombination reconstituting a functional beta-lactamase gene.

FIG. 10 schematically portrays pBR322-Sfi-2Bla-Sfi and in vivointraplasmidic recombination via direct repeats, as well as the rate ofgeneration of ampicillin-resistant colonies by intraplasmidicrecombination reconstituting a functional beta-lactamase gene.

FIG. 11 illustrates the method for testing the efficiency of multiplerounds of homologous recombination after the introduction ofpolynucleotide fragments into cells for the generation of recombinantproteins.

FIG. 12 schematically portrays generation of a library of vectors byshuffling cassettes at the following loci: promoter, leader peptide,terminator, selectable drug resistance gene, and origin of replication.The multiple parallel lines at each locus represents the multiplicity ofcassettes for that cassette.

FIG. 13 schematically shows some examples of cassettes suitable atvarious loci for constructing prokaryotic vector libraries by shuffling.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention relates to a method for nucleic acid moleculereassembly after random fragmentation and its application to mutagenesisof DNA sequences. Also described is a method for the production ofnucleic acid fragments encoding mutant proteins having enhancedbiological activity. In particular, the present invention also relatesto a method of repeated cycles of mutagenesis, nucleic acid shufflingand selection which allow for the creation of mutant proteins havingenhanced biological activity.

The present invention is directed to a method for generating a verylarge library of DNA, RNA or protein mutants. This method has particularadvantages in the generation of related DNA fragments from which thedesired nucleic acid fragment(s) may be selected. In particular thepresent invention also relates to a method of repeated cycles ofmutagenesis, homologous recombination and selection which allow for thecreation of mutant proteins having enhanced biological activity.

However, prior to discussing this invention in further detail, thefollowing terms will first be defined.

Definitions

As used herein, the following terms have the following meanings:

The term "DNA reassembly" is used when recombination occurs betweenidentical sequences.

By contrast, the term "DNA shuffling" is used herein to indicaterecombination between substantially homologous but non-identicalsequences, in some embodiments DNA shuffling may involve crossover vianonhomologous recombination, such as via cre/lox and/or flp/frt systemsand the like.

The term "amplification" means that the number of copies of a nucleicacid fragment is increased.

The term "identical" or "identity" means that two nucleic acid sequenceshave the same sequence or a complementary sequence. Thus, "areas ofidentity" means that regions or areas of a nucleic acid fragment orpolynucleotide are identical or complementary to another polynucleotideor nucleic acid fragment.

The term "corresponds to" is used herein to mean that a polynucleotidesequence is homologous (i.e., is identical, not strictly evolutionarilyrelated) to all or a portion of a reference polynucleotide sequence, orthat a polypeptide sequence is identical to a reference polypeptidesequence. In contradistinction, the term "complementary to" is usedherein to mean that the complementary sequence is homologous to all or aportion of a reference polynucleotide sequence. For illustration, thenucleotide sequence "TATAC" corresponds to a reference sequence "TATAC"and is complementary to a reference sequence "GTATA".

The following terms are used to describe the sequence relationshipsbetween two or more polynucleotides: "reference sequence", "comparisonwindow", "sequence identity", "percentage of sequence identity", and"substantial identity". A "reference sequence" is a defined sequenceused as a basis for a sequence comparison; a reference sequence may be asubset of a larger sequence, for example, as a segment of a full-lengthcDNA or gene sequence given in a sequence listing, such as apolynucleotide sequence of FIG. 1 or FIG. 2(b), or may comprise acomplete cDNA or gene sequence. Generally, a reference sequence is atleast 20 nucleotides in length, frequently at least 25 nucleotides inlength, and often at least 50 nucleotides in length. Since twopolynucleotides may each (1) comprise a sequence (i.e., a portion of thecomplete polynucleotide sequence) that is similar between the twopolynucleotides, and (2) may further comprise a sequence that isdivergent between the two polynucleotides, sequence comparisons betweentwo (or more) polynucleotides are typically performed by comparingsequences of the two polynucleotides over a "comparison window" toidentify and compare local regions of sequence similarity.

A "comparison window", as used herein, refers to a conceptual segment ofat least 20 contiguous nucleotide positions wherein a polynucleotidesequence may be compared to a reference sequence of at least 20contiguous nucleotides and wherein the portion of the polynucleotidesequence in the comparison window may comprise additions or deletions(i.e., gaps) of 20 percent or less as compared to the reference sequence(which does not comprise additions or deletions) for optimal alignmentof the two sequences. Optimal alignment of sequences for aligning acomparison window may be conducted by the local homology algorithm ofSmith and Waterman (1981) Adv. Appl. Math. 2: 482, by the homologyalignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443, by the search for similarity method of Pearson and Lipman (1988)Proc. Natl. Acad. Sci. (U.S.A.) 85: 2444, by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package Release 7.0, Genetics ComputerGroup, 575 Science Dr., Madison, Wis.), or by inspection, and the bestalignment (i.e., resulting in the highest percentage of homology overthe comparison window) generated by the various methods is selected.

The term "sequence identity" means that two polynucleotide sequences areidentical (i.e., on a nucleotide-by-nucleotide basis) over the window ofcomparison. The term "percentage of sequence identity" is calculated bycomparing two optimally aligned sequences over the window of comparison,determining the number of positions at which the identical nucleic acidbase (e.g., A, T, C, G, U, or I) occurs in both sequences to yield thenumber of matched positions, dividing the number of matched positions bythe total number of positions in the window of comparison (i.e., thewindow size), and multiplying the result by 100 to yield the percentageof sequence identity. The terms "substantial identity" as used hereindenotes a characteristic of a polynucleotide sequence, wherein thepolynucleotide comprises a sequence that has at least 80 percentsequence identity, preferably at least 85 percent identity and often 90to 95 percent sequence identity, more usually at least 99 percentsequence identity as compared to a reference sequence over a comparisonwindow of at least 20 nucleotide positions, frequently over a window ofat least 25-50 nucleotides, wherein the percentage of sequence identityis calculated by comparing the reference sequence to the polynucleotidesequence which may include deletions or additions which total 20 percentor less of the reference sequence over the window of comparison.

Conservative amino acid substitutions refer to the interchangeability ofresidues having similar side chains. For example, a group of amino acidshaving aliphatic side chains is glycine, alanine, valine, leucine, andisoleucine; a group of amino acids having aliphatic-hydroxyl side chainsis serine and threonine; a group of amino acids having amide-containingside chains is asparagine and glutamine; a group of amino acids havingaromatic side chains is phenylalanine, tyrosine, and tryptophan; a groupof amino acids having basic side chains is lysine, arginine, andhistidine; and a group of amino acids having sulfur-containing sidechains is cysteine and methionine. Preferred conservative amino acidssubstitution groups are: valine-leucine-isoleucine,phenylalanine-tyrosine, lysine-arginine, alanine-valine, andasparagine-glutamine.

The term "homologous" or "homeologous" means that one single-strandednucleic acid sequence may hybridize to a complementary single-strandednucleic acid sequence. The degree of hybridization may depend on anumber of factors including the amount of identity between the sequencesand the hybridization conditions such as temperature and saltconcentration as discussed later. Preferably the region of identity isgreater than about 5 bp, more preferably the region of identity isgreater than 10 bp.

The term "heterologous" means that one single-stranded nucleic acidsequence is unable to hybridize to another single-stranded nucleic acidsequence or its complement. Thus areas of heterology means that nucleicacid fragments or polynucleotides have areas or regions in the sequencewhich are unable to hybridize to another nucleic acid or polynucleotide.Such regions or areas are, for example, areas of mutations.

The term "cognate" as used herein refers to a gene sequence that isevolutionarily and functionally related between species. For example butnot limitation, in the human genome, the human CD4 gene is the cognategene to the mouse CD4 gene, since the sequences and structures of thesetwo genes indicate that they are highly homologous and both genes encodea protein which functions in signaling T cell activation through MHCclass II-restricted antigen recognition.

The term "wild-type" means that the nucleic acid fragment does notcomprise any mutations. A "wild-type" protein means that the proteinwill be active at a level of activity found in nature and will comprisethe amino acid sequence found in nature.

The term "related polynucleotides" means that regions or areas of thepolynucleotides are identical and regions or areas of thepolynucleotides are heterologous.

The term "chimeric polynucleotide" means that the polynucleotidecomprises regions which are wild-type and regions which are mutated. Itmay also mean that the polynucleotide comprises wild-type regions fromone polynucleotide and wild-type regions from another relatedpolynucleotide.

The term "cleaving" means digesting the polynucleotide with enzymes orbreaking the polynucleotide.

The term "population" as used herein means a collection of componentssuch as polynucleotides, nucleic acid fragments or proteins. A "mixedpopulation" means a collection of components which belong to the samefamily of nucleic acids or proteins (i.e. are related) but which differin their sequence (i.e. are not identical) and hence in their biologicalactivity.

The term "specific nucleic acid fragment" means a nucleic acid fragmenthaving certain end points and having a certain nucleic acid sequence.Two nucleic acid fragments wherein one nucleic acid fragment has theidentical sequence as a portion of the second nucleic acid fragment butdifferent ends comprise two different specific nucleic acid fragments.

The term "mutations" means changes in the sequence of a wild-typenucleic acid sequence or changes in the sequence of a peptide. Suchmutations may be point mutations such as transitions or transversions.The mutations may be deletions, insertions or duplications.

In the polypeptide notation used herein, the lefthand direction is theamino terminal direction and the righthand direction is thecarboxy-terminal direction, in accordance with standard usage andconvention. Similarly, unless specified otherwise, the lefthand end ofsingle-stranded polynucleotide sequences is the 5' end; the lefthanddirection of double-stranded polynucleotide sequences is referred to asthe 5' direction. The direction of 5' to 3' addition of nascent RNAtranscripts is referred to as the transcription direction; sequenceregions on the DNA strand having the same sequence as the RNA and whichare 5' to the 5' end of the RNA transcript are referred to as "upstreamsequences"; sequence regions on the DNA strand having the same sequenceas the RNA and which are 3' to the 3' end of the coding RNA transcriptare referred to as "downstream sequences".

The term "naturally-occurring" as used herein as applied to an objectrefers to the fact that an object can be found in nature. For example, apolypeptide or polynucleotide sequence that is present in an organism(including viruses) that can be isolated from a source in nature andwhich has not been intentionally modified by man in the laboratory isnaturally-occurring. Generally, the term naturally-occurring refers toan object as present in a non-pathological (undiseased) individual, suchas would be typical for the species.

The term "agent" is used herein to denote a chemical compound, a mixtureof chemical compounds, an array of spatially localized compounds (e.g.,a VLSIPS peptide array, polynucleotide array, and/or combinatorial smallmolecule array), a biological macromolecule, a bacteriophage peptidedisplay library, a bacteriophage antibody (e.g., scFv) display library,a polysome peptide display library, or an extract made from biologicalmaterials such as bacteria, plants, fungi, or animal (particularlymammalian) cells or tissues. Agents are evaluated for potential activityas antineoplastics, anti-inflammatories, or apoptosis modulators byinclusion in screening assays described hereinbelow. Agents areevaluated for potential activity as specific protein interactioninhibitors (i.e., an agent which selectively inhibits a bindinginteraction between two predetermined polypeptides but which does notsubstantially interfere with cell viability) by inclusion in screeningassays described hereinbelow.

As used herein, "substantially pure" means an object species is thepredominant species present (i.e., on a molar basis it is more abundantthan any other individual macromolecular species in the composition),and preferably a substantially purified fraction is a compositionwherein the object species comprises at least about 50 percent (on amolar basis) of all macromolecular species present. Generally, asubstantially pure composition will comprise more than about 80 to 90percent of all macromolecular species present in the composition. Mostpreferably, the object species is purified to essential homogeneity(contaminant species cannot be detected in the composition byconventional detection methods) wherein the composition consistsessentially of a single macromolecular species. Solvent species, smallmolecules (<500 Daltons), and elemental ion species are not consideredmacromolecular species.

As used herein the term "physiological conditions" refers totemperature, pH, ionic strength, viscosity, and like biochemicalparameters which are compatible with a viable organism, and/or whichtypically exist intracellularly in a viable cultured yeast cell ormammalian cell. For example, the intracellular conditions in a yeastcell grown under typical laboratory culture conditions are physiologicalconditions. Suitable in vitro reaction conditions for in vitrotranscription cocktails are generally physiological conditions. Ingeneral, in vitro physiological conditions comprise 50-200 mM NaCl orKCl, pH 6.5-8.5, 20°-45° C. and 0.001-10 mM divalent cation (e.g., Mg⁺⁺,Ca⁺⁺); preferably about 150 mM NaCl or KCl, pH 7.2-7.6, 5 mM divalentcation, and often include 0.01-1.0 percent nonspecific protein (e.g.,BSA). A non-ionic detergent (Tween, NP-40, Triton X-100) can often bepresent, usually at about 0.001 to 2%, typically 0.05-0.2% (v/v).Particular aqueous conditions may be selected by the practitioneraccording to conventional methods. For general guidance, the followingbuffered aqueous conditions may be applicable: 10-250 mM NaCl, 5-50 mMTris HCl, pH 5-8, with optional addition of divalent cation(s) and/ormetal chelators and/or nonionic detergents and/or membrane fractionsand/or antifoam agents and/or scintillants.

Specific hybridization is defined herein as the formation of hybridsbetween a first polynucleotide and a second polynucleotide (e.g., apolynucleotide having a distinct but substantially identical sequence tothe first polynucleotide), wherein the first polynucleotidepreferentially hybridizes to the second polynucleotide under stringenthybridization conditions wherein substantially unrelated polynucleotidesequences do not form hybrids in the mixture.

As used herein, the term "single-chain antibody" refers to a polypeptidecomprising a V_(H) domain and a V_(L) domain in polypeptide linkage,generally linked via a spacer peptide (e.g., Gly-Gly-Gly-Gly-Ser!_(x)),and which may comprise additional amino acid sequences at the amino-and/or carboxy- termini. For example, a single-chain antibody maycomprise a tether segment for linking to the encoding polynucleotide. Asan example, a scFv is a single-chain antibody. Single-chain antibodiesare generally proteins consisting of one or more polypeptide segments ofat least 10 contiguous amino acids substantially encoded by genes of theimmunoglobulin superfamily (e.g., see The Immunoglobulin GeneSuperfamily, A. F. Williams and A. N. Barclay, in Immunoglobulin Genes,T. Honjo, F. W. Alt, and T. H. Rabbitts, eds., (1989) Academic Press:San Diego, CA, pp.361-387, which is incorporated herein by reference),most frequently encoded by a rodent, non-human primate, avian, porcine,bovine, ovine, goat, or human heavy chain or light chain gene sequence.A functional single-chain antibody generally contains a sufficientportion of an immunoglobulin superfamily gene product so as to retainthe property of binding to a specific target molecule, typically areceptor or antigen (epitope).

As used herein, the term "complementarity-determining region" and "CDR"refer to the art-recognized term as exemplified by the Kabat and ChothiaCDR definitions also generally known as hypervariable regions orhypervariable loops (Chothia and Lesk (1987) J. Mol. Biol. 196: 901;Chothia et al. (1989) Nature 342: 877; E. A. Kabat et al., Sequences ofProteins of Immunological Interest (National Institutes of Health,Bethesda, Md.) (1987); and Tramontano et al. (1990) J. Mol. Biol. 215:175). Variable region domains typically comprise the amino-terminalapproximately 105-115 amino acids of a naturally-occurringimmunoglobulin chain (e.g., amino acids 1-110), although variabledomains somewhat shorter or longer are also suitable for formingsingle-chain antibodies.

An immunoglobulin light or heavy chain variable region consists of a"framework" region interrupted by three hypervariable regions, alsocalled CDR's. The extent of the framework region and CDR's have beenprecisely defined (see, "Sequences of Proteins of ImmunologicalInterest," E. Kabat et al., 4th Ed., U.S. Department of Health and HumanServices, Bethesda, Md. (1987)). The sequences of the framework regionsof different light or heavy chains are relatively conserved within aspecies. As used herein, a "human framework region" is a frameworkregion that is substantially identical (about 85% or more, usually90-95% or more) to the framework region of a naturally occurring humanimmunoglobulin. The framework region of an antibody, that is thecombined framework regions of the constituent light and heavy chains,serves to position and align the CDR's. The CDR's are primarilyresponsible for binding to an epitope of an antigen.

As used herein, the term "variable segment" refers to a portion of anascent peptide which comprises a random, pseudorandom, or definedkernal sequence. A variable segment can comprise both variant andinvariant residue positions, and the degree of residue variation at avariant residue position may be limited; both options are selected atthe discretion of the practitioner. Typically, variable segments areabout 5 to 20 amino acid residues in length (e.g., 8 to 10), althoughvariable segments may be longer and may comprise antibody portions orreceptor proteins, such as an antibody fragment, a nucleic acid bindingprotein, a receptor protein, and the like.

As used herein, "random peptide sequence" refers to an amino acidsequence composed of two or more amino acid monomers and constructed bya stochastic or random process. A random peptide can include frameworkor scaffolding motifs, which may comprise invariant sequences.

As used herein "random peptide library" refers to a set ofpolynucleotide sequences that encodes a set of random peptides, and tothe set of random peptides encoded by those polynucleotide sequences, aswell as the fusion proteins containing those random peptides.

As used herein, the term "pseudorandom" refers to a set of sequencesthat have limited variability, so that for example the degree of residuevariability at one position is different than the degree of residuevariability at another position, but any pseudorandom position isallowed some degree of residue variation, however circumscribed.

As used herein, the term "defined sequence framework" refers to a set ofdefined sequences that are selected on a nonrandom basis, generally onthe basis of experimental data or structural data; for example, adefined sequence framework may comprise a set of amino acid sequencesthat are predicted to form a β-sheet structure or may comprise a leucinezipper heptad repeat motif, a zinc-finger domain, among othervariations. A "defined sequence kernal" is a set of sequences whichencompass a limited scope of variability. Whereas (1) a completelyrandom 10-mer sequence of the 20 conventional amino acids can be any of(20)¹⁰ sequences, and (2) a pseudorandom 10-mer sequence of the 20conventional amino acids can be any of (20)¹⁰ sequences but will exhibita bias for certain residues at certain positions and/or overall, (3) adefined sequence kernal is a subset of sequences which is less that themaximum number of potential sequences if each residue position wasallowed to be any of the allowable 20 conventional amino acids (and/orallowable unconventional amino/imino acids). A defined sequence kernalgenerally comprises variant and invariant residue positions and/orcomprises variant residue positions which can comprise a residueselected from a defined subset of amino acid residues), and the like,either segmentally or over the entire length of the individual selectedlibrary member sequence. Defined sequence kernals can refer to eitheramino acid sequences or polynucleotide sequences. For illustration andnot limitation, the sequences (NNK)₁₀ and (NNM)₁₀ where N represents A,T, G, or C; K represents G or T; and M represents A or. C, are definedsequence kernals.

As used herein "epitope" refers to that portion of an antigen or othermacromolecule capable of forming a binding interaction that interactswith the variable region binding pocket of an antibody. Typically, suchbinding interaction is manifested as an intermolecular contact with oneor more amino acid residues of a CDR.

As used herein, "receptor" refers to a molecule that has an affinity fora given ligand. Receptors can be naturally occurring or syntheticmolecules. Receptors can be employed in an unaltered state or asaggregates with other species. Receptors can be attached, covalently ornoncovalently, to a binding member, either directly or via a specificbinding substance. Examples of receptors include, but are not limitedto, antibodies, including monoclonal antibodies and antisera reactivewith specific antigenic determinants (such as on viruses, cells, orother materials), cell membrane receptors, complex carbohydrates andglycoproteins, enzymes, and hormone receptors.

As used herein "ligand" refers to a molecule, such as a random peptideor variable segment sequence, that is recognized by a particularreceptor. As one of skill in the art will recognize, a molecule (ormacromolecular complex) can be both a receptor and a ligand. In general,the binding partner having a smaller molecular weight is referred to asthe ligand and the binding partner having a greater molecular weight isreferred to as a receptor.

As used herein, "linker" or "spacer" refers to a molecule or group ofmolecules that connects two molecules, such as a DNA binding protein anda random peptide, and serves to place the two molecules in a preferredconfiguration, e.g., so that the random peptide can bind to a receptorwith minimal steric hindrance from the DNA binding protein.

As used herein, the term "operably linked" refers to a linkage ofpolynucleotide elements in a functional relationship. A nucleic acid is"operably linked" when it is placed into a functional relationship withanother nucleic acid sequence. For instance, a promoter or enhancer isoperably linked to a coding sequence if it affects the transcription ofthe coding sequence. Operably linked means that the DNA sequences beinglinked are typically contiguous and, where necessary to join two proteincoding regions, contiguous and in reading frame.

Methodology

Nucleic acid shuffling is a method for in vitro or in vivo homologousrecombination of pools of nucleic acid fragments or polynucleotides.Mixtures of related nucleic acid sequences or polynucleotides arerandomly fragmented, and reassembled to yield a library or mixedpopulation of recombinant nucleic acid molecules or polynucleotides.

In contrast to cassette mutagenesis, only shuffling and error-prone PCRallow one to mutate a pool of sequences blindly (without sequenceinformation other than primers).

The advantage of the mutagenic shuffling of this invention overerror-prone PCR alone for repeated selection can best be explained withan example from antibody engineering. In FIG. 1 is shown a schematicdiagram of DNA shuffling as described herein. The initial library canconsist of related sequences of diverse origin (i.e. antibodies fromnaive mRNA) or can be derived by any type of mutagenesis (includingshuffling) of a single antibody gene. A collection of selectedcomplementarity determining regions ("CDRs") is obtained after the firstround of affinity selection (FIG. 1). In the diagram the thick CDRsconfer onto the antibody molecule increased affinity for the antigen.Shuffling allows the free combinatorial association of all of the CDR1swith all of the CDR2s with all of the CDR3s, etc. (FIG. 1).

This method differs from PCR, in that it is an inverse chain reaction.In PCR, the number of polymerase start sites and the number of moleculesgrows exponentially. However, the sequence of the polymerase start sitesand the sequence of the molecules remains essentially the same. Incontrast, in nucleic acid reassembly or shuffling of random fragmentsthe number of start sites and the number (but not size) of the randomfragments decreases over time. For fragments derived from whole plasmidsthe theoretical endpoint is a single, large concatemeric molecule.

Since cross-overs occur at regions of homology, recombination willprimarily occur between members of the same sequence family. Thisdiscourages combinations of CDRs that are grossly incompatible (eg.directed against different epitopes of the same antigen). It iscontemplated that multiple families of sequences can be shuffled in thesame reaction. Further, shuffling conserves the relative order, suchthat, for example, CDR1 will not be found in the position of CDR2.

Rare shufflants will contain a large number of the best (eg. highestaffinity) CDRs and these rare shufflants may be selected based on theirsuperior affinity (FIG. 1).

CDRs from a pool of 100 different selected antibody sequences can bepermutated in up to 100⁶ different ways. This large number ofpermutations cannot be represented in a single library of DNA sequences.Accordingly, it is contemplated that multiple cycles of DNA shufflingand selection may be required depending on the length of the sequenceand the sequence diversity desired.

Error-prone PCR, in contrast, keeps all the selected CDRs in the samerelative sequence (FIG. 1), generating a much smaller mutant cloud.

The template polynucleotide which may be used in the methods of thisinvention may be DNA or RNA. It may be of various lengths depending onthe size of the gene or DNA fragment to be recombined or reassembled.Preferably the template polynucleotide is from 50 bp to 50 kb. It iscontemplated that entire vectors containing the nucleic acid encodingthe protein of interest can be used in the methods of this invention,and in fact have been successfully used.

The template polynucleotide may be obtained by amplification using thePCR reaction (U.S. Pat. Nos. 4,683,202 and 4,683,195) or otheramplification or cloning methods. However, the removal of free primersfrom the PCR product before fragmentation provides a more efficientresult. Failure to adequately remove the primers can lead to a lowfrequency of crossover clones.

The template polynucleotide often should be double-stranded. Adouble-stranded nucleic acid molecule is required to ensure that regionsof the resulting single-stranded nucleic acid fragments arecomplementary to each other and thus can hybridize to form adouble-stranded molecule.

It is contemplated that single-stranded or double-stranded nucleic acidfragments having regions of identity to the template polynucleotide andregions of heterology to the template polynucleotide may be added to thetemplate polynucleotide at this step. It is also contemplated that twodifferent but related polynucleotide templates can be mixed at thisstep.

The double-stranded polynucleotide template and any added double-orsingle-stranded fragments are randomly digested into fragments of fromabout 5 bp to 5 kb or more. Preferably the size of the random fragmentsis from about 10 bp to 1000 bp, more preferably the size of the DNAfragments is from about 20 bp to 500 bp.

Alternatively, it is also contemplated that double-stranded nucleic acidhaving multiple nicks may be used in the methods of this invention. Anick is a break in one strand of the double-stranded nucleic acid. Thedistance between such nicks is preferably 5 bp to 5 kb, more preferablybetween 10 bp to 1000 bp.

The nucleic acid fragment may be digested by a number of differentmethods. The nucleic acid fragment may be digested with a nuclease, suchas DNAseI or RNAse. The nucleic acid may be randomly sheared by themethod of sonication or by passage through a tube having a smallorifice.

It is also contemplated that the nucleic acid may also be partiallydigested with one or more restriction enzymes, such that certain pointsof cross-over may be retained statistically.

The concentration of any one specific nucleic acid fragment will not begreater than 1% by weight of the total nucleic acid, more preferably theconcentration of any one specific nucleic acid sequence will not begreater than 0.1% by weight of the total nucleic acid.

The number of different specific nucleic acid fragments in the mixturewill be at least about 100, preferably at least about 500, and morepreferably at least about 1000.

At this step single-stranded or double-stranded nucleic acid fragments,either synthetic or natural, may be added to the random double-strandednucleic acid fragments in order to increase the heterogeneity of themixture of nucleic acid fragments.

It is also contemplated that populations of double-stranded randomlybroken nucleic acid fragments may be mixed or combined at this step.

Where insertion of mutations into the template polynucleotide isdesired, single-stranded or double-stranded nucleic acid fragmentshaving a region of identity to the template polynucleotide and a regionof heterology to the template polynucleotide may be added in a 20 foldexcess by weight as compared to the total nucleic acid, more preferablythe single-stranded nucleic acid fragments may be added in a 10 foldexcess by weight as compared to the total nucleic acid.

Where a mixture of different but related template polynucleotides isdesired, populations of nucleic acid fragments from each of thetemplates may be combined at a ratio of less than about 1:100, morepreferably the ratio is less than about 1:40. For example, a backcrossof the wild-type polynucleotide with a population of mutatedpolynucleotide may be desired to eliminate neutral mutations (e.g.,mutations yielding an insubstantial alteration in the phenotypicproperty being selected for). In such an example, the ratio of randomlydigested wild-type polynucleotide fragments which may be added to therandomly digested mutant polynucleotide fragments is approximately 1:1to about 100:1, and more preferably from 1:1 to 40:1.

The mixed population of random nucleic acid fragments are denatured toform single-stranded nucleic acid fragments and then reannealed. Onlythose single-stranded nucleic acid fragments having regions of homologywith other single-stranded nucleic acid fragments will reanneal.

The random nucleic acid fragments may be denatured by heating. Oneskilled in the art could determine the conditions necessary tocompletely denature the double stranded nucleic acid. Preferably thetemperature is from 80° C. to 100° C., more preferably the temperatureis from 90° C. to 96° C. Other methods which may be used to denature thenucleic acid fragments include pressure (36) and pH.

The nucleic acid fragments may be reannealed by cooling. Preferably thetemperature is from 20° C. to 75° C., more preferably the temperature isfrom 40° C. to 65° C. If a high frequency of crossovers is needed basedon an average of only 4 consecutive bases of homology, recombination canbe forced by using a low annealing temperature, although the processbecomes more difficult. The degree of renaturation which occurs willdepend on the degree of homology between the population ofsingle-stranded nucleic acid fragments.

Renaturation can be accelerated by the addition of polyethylene glycol("PEG") or salt. The salt concentration is preferably from 0 mM to 200mM, more preferably the salt concentration is from 10 mM to 100 mM. Thesalt may be KCl or NaCl. The concentration of PEG is preferably from 0%to 20%, more preferably from 5% to 10%.

The annealed nucleic acid fragments are next incubated in the presenceof a nucleic acid polymerase and dNTP's (i.e. dATP, dCTP, dGTP anddTTP). The nucleic acid polymerase may be the Klenow fragment, the Taqpolymerase or any other DNA polymerase known in the art.

The approach to be used for the assembly depends on the minimum degreeof homology that should still yield crossovers. If the areas of identityare large, Taq polymerase can be used with an annealing temperature ofbetween 45°-65° C. If the areas of identity are small, Klenow polymerasecan be used with an annealing temperature of between 20°-30° C. Oneskilled in the art could vary the temperature of annealing to increasethe number of cross-overs achieved.

The polymerase may be added to the random nucleic acid fragments priorto annealing, simultaneously with annealing or after annealing.

The cycle of denaturation, renaturation and incubation in the presenceof polymerase is referred to herein as shuffling or reassembly of thenucleic acid. This cycle is repeated for a desired number of times.Preferably the cycle is repeated from 2 to 50 times, more preferably thesequence is repeated from 10 to 40 times.

The resulting nucleic acid is a larger double-stranded polynucleotide offrom about 50 bp to about 100 kb, preferably the larger polynucleotideis from 500 bp to 50 kb.

This larger polynucleotide fragment may contain a number of copies of anucleic acid fragment having the same size as the templatepolynucleotide in tandem. This concatemeric fragment is then digestedinto single copies of the template polynucleotide. The result will be apopulation of nucleic acid fragments of approximately the same size asthe template polynucleotide. The population will be a mixed populationwhere single or double-stranded nucleic acid fragments having an area ofidentity and an area of heterology have been added to the templatepolynucleotide prior to shuffling.

These fragment are then cloned into the appropriate vector and theligation mixture used to transform bacteria.

It is contemplated that the single nucleic acid fragments may beobtained from the larger concatemeric nucleic acid fragment byamplification of the single nucleic acid fragments prior to cloning by avariety of methods including PCR (U.S. Pat. Nos. 4,683,195 and4,683,202) rather than by digestion of the concatemer.

The vector used for cloning is not critical provided that it will accepta DNA fragment of the desired size. If expression of the DNA fragment isdesired, the cloning vehicle should further comprise transcription andtranslation signals next to the site of insertion of the DNA fragment toallow expression of the DNA fragment in the host cell. Preferred vectorsinclude the pUC series and the pBR series of plasmids.

The resulting bacterial population will include a number of recombinantDNA fragments having random mutations. This mixed population may betested to identify the desired recombinant nucleic acid fragment. Themethod of selection will depend on the DNA fragment desired.

For example, if a DNA fragment which encodes for a protein withincreased binding efficiency to a ligand is desired, the proteinsexpressed by each of the DNA fragments in the population or library maybe tested for their ability to bind to the ligand by methods known inthe art (i.e. panning, affinity chromatography). If a DNA fragment whichencodes for a protein with increased drug resistance is desired, theproteins expressed by each of the DNA fragments in the population orlibrary may be tested for their ability to confer drug resistance to thehost organism. One skilled in the art, given knowledge of the desiredprotein, could readily test the population to identify DNA fragmentswhich confer the desired properties onto the protein.

It is contemplated that one skilled in the art could use a phage displaysystem in which fragments of the protein are expressed as fusionproteins on the phage surface (Pharmacia, Milwaukee, Wis.). Therecombinant DNA molecules are cloned into the phage DNA at a site whichresults in the transcription of a fusion protein a portion of which isencoded by the recombinant DNA molecule. The phage containing therecombinant nucleic acid molecule undergoes replication andtranscription in the cell. The leader sequence of the fusion proteindirects the transport of the fusion protein to the tip of the phageparticle. Thus the fusion protein which is partially encoded by therecombinant DNA molecule is displayed on the phage particle fordetection and selection by the methods described above.

It is further contemplated that a number of cycles of nucleic acidshuffling may be conducted with nucleic acid fragments from asubpopulation of the first population, which subpopulation contains DNAencoding the desired recombinant protein. In this manner, proteins witheven higher binding affinities or enzymatic activity could be achieved.

It is also contemplated that a number of cycles of nucleic acidshuffling may be conducted with a mixture of wild-type nucleic acidfragments and a subpopulation of nucleic acid from the first orsubsequent rounds of nucleic acid shuffling in order to remove anysilent mutations from the subpopulation.

Any source of nucleic acid, in purified form can be utilized as thestarting nucleic acid. Thus the process may employ DNA or RNA includingmessenger RNA, which DNA or RNA may be single or double stranded. Inaddition, a DNA-RNA hybrid which contains one strand of each may beutilized. The nucleic acid sequence may be of various lengths dependingon the size of the nucleic acid sequence to be mutated. Preferably thespecific nucleic acid sequence is from 50 to 50,000 base pairs. It iscontemplated that entire vectors containing the nucleic acid encodingthe protein of interest may be used in the methods of this invention.

The nucleic acid may be obtained from any source, for example, fromplasmids such a pBR322, from cloned DNA or RNA or from natural DNA orRNA from any source including bacteria, yeast, viruses and higherorganisms such as plants or animals. DNA or RNA may be extracted fromblood or tissue material. The template polynucleotide may be obtained byamplification using the polynucleotide chain reaction (PCR) (U.S. Pat.Nos. 4,683,202 and 4,683,195). Alternatively, the polynucleotide may bepresent in a vector present in a cell and sufficient nucleic acid may beobtained by culturing the cell and extracting the nucleic acid from thecell by methods known in the art.

Any specific nucleic acid sequence can be used to produce the populationof mutants by the present process. It is only necessary that a smallpopulation of mutant sequences of the specific nucleic acid sequenceexist or be created prior to the present process.

The initial small population of the specific nucleic acid sequenceshaving mutations may be created by a number of different methods.Mutations may be created by error-prone PCR. Error-prone PCR useslow-fidelity polymerization conditions to introduce a low level of pointmutations randomly over a long sequence. Alternatively, mutations can beintroduced into the template polynucleotide by oligonucleotide-directedmutagenesis. In oligonucleotide-directed mutagenesis, a short sequenceof the polynucleotide is removed from the polynucleotide usingrestriction enzyme digestion and is replaced with a syntheticpolynucleotide in which various bases have been altered from theoriginal sequence. The polynucleotide sequence can also be altered bychemical mutagenesis. Chemical mutagens include, for example, sodiumbisulfite, nitrous acid, hydroxylamine, hydrazine or formic acid. Otheragents which are analogues of nucleotide precursors includenitrosoguanidine, 5-bromouracil, 2-aminopurine, or acridine. Generally,these agents are added to the PCR reaction in place of the nucleotideprecursor thereby mutating the sequence. Intercalating agents such asproflavine, acriflavine, quinacrine and the like can also be used.Random mutagenesis of the polynucleotide sequence can also be achievedby irradiation with X-rays or ultraviolet light. Generally, plasmid DNAor DNA fragments so mutagenized are introduced into E. coli andpropagated as a pool or library of mutant plasmids.

Alternatively the small mixed population of specific nucleic acids maybe found in nature in that they may consist of different alleles of thesame gene or the same gene from different related species (i.e., cognategenes). Alternatively, they may be related DNA sequences found withinone species, for example, the immunoglobulin genes.

Once the mixed population of the specific nucleic acid sequences isgenerated, the polynucleotides can be used directly or inserted into anappropriate cloning vector, using techniques well-known in the art.

The choice of vector depends on the size of the polynucleotide sequenceand the host cell to be employed in the methods of this invention. Thetemplates of this invention may be plasmids, phages, cosmids, phagemids,viruses (e.g., retroviruses, parainfluenzavirus, herpesviruses,reoviruses, paramyxoviruses, and the like), or selected portions thereof(e.g., coat protein, spike glycoprotein, capsid protein). For example,cosmids and phagemids are preferred where the specific nucleic acidsequence to be mutated is larger because these vectors are able tostably propagate large nucleic acid fragments.

If the mixed population of the specific nucleic acid sequence is clonedinto a vector it can be clonally amplified by inserting each vector intoa host cell and allowing the host cell to amplify the vector. This isreferred to as clonal amplification because while the absolute number ofnucleic acid sequences increases, the number of mutants does notincrease. Utility

The DNA shuffling method of this invention can be performed blindly on apool of unknown sequences. By adding to the reassembly mixtureoligonucleotides (with ends that are homologous to the sequences beingreassembled) any sequence mixture can be incorporated at any specificposition into another sequence mixture. Thus, it is contemplated thatmixtures of synthetic oligonucleotides, PCR fragments or even wholegenes can be mixed into another sequence library at defined positions.The insertion of one sequence (mixture) is independent from theinsertion of a sequence in another part of the template. Thus, thedegree of recombination, the homology required, and the diversity of thelibrary can be independently and simultaneously varied along the lengthof the reassembled DNA.

This approach of mixing two genes may be useful for the humanization ofantibodies from murine hybridomas. The approach of mixing two genes orinserting mutant sequences into genes may be useful for anytherapeutically used protein, for example, interleukin I, antibodies,tPA, growth hormone, etc. The approach may also be useful in any nucleicacid for example, promoters or introns or 3' untranslated region or 5'untranslated regions of genes to increase expression or alterspecificity of expression of proteins. The approach may also be used tomutate ribozymes or aptamers.

Shuffling requires the presence of homologous regions separating regionsof diversity. Scaffold-like protein structures may be particularlysuitable for shuffling. The conserved scaffold determines the overallfolding by self-association, while displaying relatively unrestrictedloops that mediate the specific binding. Examples of such scaffolds arethe immunoglobulin beta-barrel, and the four-helix bundle (24). Thisshuffling can be used to create scaffold-like proteins with variouscombinations of mutated sequences for binding.

In Vitro Shuffling

The equivalents of some standard genetic matings may also be performedby shuffling in vitro. For example, a `molecular backcross` can beperformed by repeated mixing of the mutant's nucleic acid with thewild-type nucleic acid while selecting for the mutations of interest. Asin traditional breeding, this approach can be used to combine phenotypesfrom different sources into a background of choice. It is useful, forexample, for the removal of neutral mutations that affect unselectedcharacteristics (i.e. immunogenicity). Thus it can be useful todetermine which mutations in a protein are involved in the enhancedbiological activity and which are not, an advantage which cannot beachieved by error-prone mutagenesis or cassette mutagenesis methods.

Large, functional genes can be assembled correctly from a mixture ofsmall random fragments. This reaction may be of use for the reassemblyof genes from the highly fragmented DNA of fossils (25). In additionrandom nucleic acid fragments from fossils may be combined with nucleicacid fragments from similar genes from related species.

It is also contemplated that the method of this invention can be usedfor the in vitro amplification of a whole genome from a single cell asis needed for a variety of research and diagnostic applications. DNAamplification by PCR is in practice limited to a length of about 40 kb.Amplification of a whole genome such as that of E. coli (5,000 kb) byPCR would require about 250 primers yielding 125 forty kb fragments.This approach is not practical due to the unavailability of sufficientsequence data. On the other hand, random digestion of the genome withDNAseI, followed by gel purification of small fragments will provide amultitude of possible primers. Use of this mix of random small fragmentsas primers in a PCR reaction alone or with the whole genome as thetemplate should result in an inverse chain reaction with the theoreticalendpoint of a single concatemer containing many copies of the genome.

100 fold amplification in the copy number and an average fragment sizeof greater than 50 kb may be obtained when only random fragments areused (see Example 2). It is thought that the larger concatemer isgenerated by overlap of many smaller fragments. The quality of specificPCR products obtained using synthetic primers will be indistinguishablefrom the product obtained from unamplified DNA. It is expected that thisapproach will be useful for the mapping of genomes.

The polynucleotide to be shuffled can be fragmented randomly ornon-randomly, at the discretion of the practitioner.

In Vivo Shuffling

In an embodiment of in vivo shuffling, the mixed population of thespecific nucleic acid sequence is introduced into bacterial oreukaryotic cells under conditions such that at least two differentnucleic acid sequences are present in each host cell. The fragments canbe introduced into the host cells by a variety of different methods. Thehost cells can be transformed with the fragments using methods known inthe art, for example treatment with calcium chloride. If the fragmentsare inserted into a phage genome, the host cell can be transfected withthe recombinant phage genome having the specific nucleic acid sequences.Alternatively, the nucleic acid sequences can be introduced into thehost cell using electroporation, transfection, lipofection, biolistics,conjugation, and the like.

In general, in this embodiment, the specific nucleic acids sequenceswill be present in vectors which are capable of stably replicating thesequence in the host cell. In addition, it is contemplated that thevectors will encode a marker gene such that host cells having the vectorcan be selected. This ensures that the mutated specific nucleic acidsequence can be recovered after introduction into the host cell.However, it is contemplated that the entire mixed population of thespecific nucleic acid sequences need not be present on a vectorsequence. Rather only a sufficient number of sequences need be clonedinto vectors to ensure that after introduction of the fragments into thehost cells each host cell contains one vector having at least onespecific nucleic acid sequence present therein. It is also contemplatedthat rather than having a subset of the population of the specificnucleic acids sequences cloned into vectors, this subset may be alreadystably integrated into the host cell.

It has been found that when two fragments which have regions of identityare inserted into the host cells homologous recombination occurs betweenthe two fragments. Such recombination between the two mutated specificnucleic acid sequences will result in the production of double or triplemutants in some situations.

It has also been found that the frequency of recombination is increasedif some of the mutated specific nucleic acid sequences are present onlinear nucleic acid molecules. Therefore, in a preferred embodiment,some of the specific nucleic acid sequences are present on linearnucleic acid fragments.

After transformation, the host cell transformants are placed underselection to identify those host cell transformants which containmutated specific nucleic acid sequences having the qualities desired.For example, if increased resistance to a particular drug is desiredthen the transformed host cells may be subjected to increasedconcentrations of the particular drug and those transformants producingmutated proteins able to confer increased drug resistance will beselected. If the enhanced ability of a particular protein to bind to areceptor is desired, then expression of the protein can be induced fromthe transformants and the resulting protein assayed in a ligand bindingassay by methods known in the art to identify that subset of the mutatedpopulation which shows enhanced binding to the ligand. Alternatively,the protein can be expressed in another system to ensure properprocessing.

Once a subset of the first recombined specific nucleic acid sequences(daughter sequences) having the desired characteristics are identified,they are then subject to a second round of recombination.

In the second cycle of recombination, the recombined specific nucleicacid sequences may be mixed with the original mutated specific nucleicacid sequences (parent sequences) and the cycle repeated as describedabove. In this way a set of second recombined specific nucleic acidssequences can be identified which have enhanced characteristics orencode for proteins having enhanced properties. This cycle can berepeated a number of times as desired.

It is also contemplated that in the second or subsequent recombinationcycle, a backcross can be performed. A molecular backcross can beperformed by mixing the desired specific nucleic acid sequences with alarge number of the wild-type sequence, such that at least one wild-typenucleic acid sequence and a mutated nucleic acid sequence are present inthe same host cell after transformation. Recombination with thewild-type specific nucleic acid sequence will eliminate those neutralmutations that may affect unselected characteristics such asimmunogenicity but not the selected characteristics.

In another embodiment of this invention, it is contemplated that duringthe first round a subset of the specific nucleic acid sequences can befragmented prior to introduction into the host cell. The size of thefragments must be large enough to contain some regions of identity withthe other sequences so as to homologously recombine with the othersequences. The size of the fragments will range from 0.03 kb to 100 kbmore preferably from 0.2 kb to 10 kb. It is also contemplated that insubsequent rounds, all of the specific nucleic acid sequences other thanthe sequences selected from the previous round may be cleaved intofragments prior to introduction into the host cells.

Fragmentation of the sequences can be accomplished by a variety ofmethod known in the art. The sequences can be randomly fragmented orfragmented at specific sites in the nucleic acid sequence. Randomfragments can be obtained by breaking the nucleic acid or exposing it toharsh physical treatment (e.g., shearing or irradiation) or harshchemical agents (e.g., by free radicals; metal ions; acid treatment todepurinate and cleave). Random fragments can also be obtained, in thecase of DNA by the use of DNase or like nuclease. The sequences can becleaved at specific sites by the use of restriction enzymes. Thefragmented sequences can be single-stranded or double-stranded. If thesequences were originally single-stranded they can be denatured withheat, chemicals or enzymes prior to insertion into the host cell. Thereaction conditions suitable for separating the strands of nucleic acidare well known in the art.

The steps of this process can be repeated indefinitely, being limitedonly by the number of possible mutants which can be achieved. After acertain number of cycles, all possible mutants will have been achievedand further cycles are redundant.

In an embodiment the same mutated template nucleic acid is repeatedlyrecombined and the resulting recombinants selected for the desiredcharacteristic.

Therefore, the initial pool or population of mutated template nucleicacid is cloned into a vector capable of replicating in a bacteria suchas E. coli. The particular vector is not essential, so long as it iscapable of autonomous replication in E. coli. In a preferred embodiment,the vector is designed to allow the expression and production of anyprotein encoded by the mutated specific nucleic acid linked to thevector. It is also preferred that the vector contain a gene encoding fora selectable marker.

The population of vectors containing the pool of mutated nucleic acidsequences is introduced into the E. coli host cells. The vector nucleicacid sequences may be introduced by transformation, transfection orinfection in the case of phage. The concentration of vectors used totransform the bacteria is such that a number of vectors is introducedinto each cell. Once present in the cell, the efficiency of homologousrecombination is such that homologous recombination occurs between thevarious vectors. This results in the generation of mutants (daughters)having a combination of mutations which differ from the original parentmutated sequences.

The host cells are then clonally replicated and selected for the markergene present on the vector. Only those cells having a plasmid will growunder the selection.

The host cells which contain a vector are then tested for the presenceof favorable mutations. Such testing may consist of placing the cellsunder selective pressure, for example, if the gene to be selected is animproved drug resistance gene. If the vector allows expression of theprotein encoded by the mutated nucleic acid sequence, then suchselection may include allowing expression of the protein so encoded,isolation of the protein and testing of the protein to determinewhether, for example, it binds with increased efficiency to the ligandof interest.

Once a particular daughter mutated nucleic acid sequence has beenidentified which confers the desired characteristics, the nucleic acidis isolated either already linked to the vector or separated from thevector. This nucleic acid is then mixed with the first or parentpopulation of nucleic acids and the cycle is repeated.

It has been shown that by this method nucleic acid sequences havingenhanced desired properties can be selected.

In an alternate embodiment, the first generation of mutants are retainedin the cells and the parental mutated sequences are added again to thecells. Accordingly, the first cycle of Embodiment I is conducted asdescribed above. However, after the daughter nucleic acid sequences areidentified, the host cells containing these sequences are retained.

The parent mutated specific nucleic acid population, either as fragmentsor cloned into the same vector is introduced into the host cells alreadycontaining the daughter nucleic acids. Recombination is allowed to occurin the cells and the next generation of recombinants, or granddaughtersare selected by the methods described above.

This cycle can be repeated a number of times until the nucleic acid orpeptide having the desired characteristics is obtained. It iscontemplated that in subsequent cycles, the population of mutatedsequences which are added to the preferred mutants may come from theparental mutants or any subsequent generation.

In an alternative embodiment, the invention provides a method ofconducting a "molecular" backcross of the obtained recombinant specificnucleic acid in order to eliminate any neutral mutations. Neutralmutations are those mutations which do not confer onto the nucleic acidor peptide the desired properties. Such mutations may however confer onthe nucleic acid or peptide undesirable characteristics. Accordingly, itis desirable to eliminate such neutral mutations. The method of thisinvention provide a means of doing so.

In this embodiment, after the mutant nucleic acid, having the desiredcharacteristics, is obtained by the methods of the embodiments, thenucleic acid, the vector having the nucleic acid or the host cellcontaining the vector and nucleic acid is isolated.

The nucleic acid or vector is then introduced into the host cell with alarge excess of the wild-type nucleic acid. The nucleic acid of themutant and the nucleic acid of the wild-type sequence are allowed torecombine. The resulting recombinants are placed under the sameselection as the mutant nucleic acid. Only those recombinants whichretained the desired characteristics will be selected. Any silentmutations which do not provide the desired characteristics will be lostthrough recombination with the wild-type DNA. This cycle can be repeateda number of times until all of the silent mutations are eliminated.

Thus the methods of this invention can be used in a molecular backcrossto eliminate unnecessary or silent mutations.

Utility

The in vivo recombination method of this invention can be performedblindly on a pool of unknown mutants or alleles of a specific nucleicacid fragment or sequence. However, it is not necessary to know theactual DNA or RNA sequence of the specific nucleic acid fragment.

The approach of using recombination within a mixed population of genescan be useful for the generation of any useful proteins, for example,interleukin I, antibodies, tPA, growth hormone, etc. This approach maybe used to generate proteins having altered specificity or activity. Theapproach may also be useful for the generation of mutant nucleic acidsequences, for example, promoter regions, introns, exons, enhancersequences, 3' untranslated regions or 5' untranslated regions of genes.Thus this approach may be used to generate genes having increased ratesof expression. This approach may also be useful in the study ofrepetitive DNA sequences. Finally, this approach may be useful to mutate:ribozymes or aptamers.

Scaffold-like regions separating regions of diversity in proteins may beparticularly suitable for the methods of this invention.l The conservedscaffold determines the overall folding by self-association, whiledisplaying relatively unrestricted loops that mediate the specificbinding. Examples of such scaffolds are the immunoglobulin beta barrel,and the four-helix bundle. The methods of this invention can be used tocreate scaffold-like proteins with various combinations of mutatedsequences for binding.

The equivalents of some standard genetic matings may also be performedby the methods of this invention. For example, a "molecular" backcrosscan be performed by repeated mixing of the mutant's nucleic acid withthe wild-type nucleic acid while selecting for the mutations ofinterest. As in traditional breeding, this approach can be used tocombine phenotypes from different sources into a background of choice.It is useful, for example, for the removal of neutral mutations thataffect unselected characteristics (i.e. immunogenicity). Thus it can beuseful to determine which mutations in a protein are involved in theenhanced biological activity and which are not.

Peptide Display Methods

The present method can be used to shuffle, by in vitro and/or in vivorecombination by any of the disclosed methods, and in any combination,polynucleotide sequences selected by peptide display methods, wherein anassociated polynucleotide encodes a displayed peptide which is screenedfor a phenotype (e.g., for affinity for a predetermined receptor(ligand).

An increasingly important aspect of biopharmaceutical drug developmentand molecular biology is the identification of peptide structures,including the primary amino acid sequences, of peptides orpeptidomimetics that interact with biological macromolecules. One methodof identifying peptides that possess a desired structure or functionalproperty, such as binding to a predetermined biological macromolecule(e.g., a receptor), involves the screening of a large library orpeptides for individual library members which possess the desiredstructure or functional property conferred by the amino acid sequence ofthe peptide.

In addition to direct chemical synthesis methods for generating peptidelibraries, several recombinant DNA methods also have been reported. Onetype involves the display of a peptide sequence, antibody, or otherprotein on the surface of a bacteriophage particle or cell. Generally,in these methods each bacteriophage particle or cell serves as anindividual library member displaying a single species of displayedpeptide in addition to the natural bacteriophage or cell proteinsequences. Each bacteriophage or cell contains the nucleotide sequenceinformation encoding the particular displayed peptide sequence; thus,the displayed peptide sequence can be ascertained by nucleotide sequencedetermination of an isolated library member.

A well-known peptide display method involves the presentation of apeptide sequence on the surface of a filamentous bacteriophage,typically as a fusion with a bacteriophage coat protein. Thebacteriophage library can be incubated with an immobilized,predetermined macromolecule or small molecule (e.g., a receptor) so thatbacteriophage particles which present a peptide sequence that binds tothe immobilized macromolecule can be differentially partitioned fromthose that do not present peptide sequences that bind to thepredetermined macromolecule. The bacteriophage particles (i.e., librarymembers) which are bound to the immobilized macromolecule are thenrecovered and replicated to amplify the selected bacteriophagesubpopulation for a subsequent round of affinity enrichment and phagereplication. After several rounds of affinity enrichment and phagereplication, the bacteriophage library members that are thus selectedare isolated and the nucleotide sequence encoding the displayed peptidesequence is determined, thereby identifying the sequence(s) of peptidesthat bind to the predetermined macromolecule (e.g., receptor). Suchmethods are further described in PCT patent publication Nos. 91/17271,91/18980, and 91/19818 and 93/08278.

The latter PCT publication describes a recombinant DNA method for thedisplay of peptide ligands that involves the production of a library offusion proteins with each fusion protein composed of a first polypeptideportion, typically comprising a variable sequence, that is available forpotential binding to a predetermined macromolecule, and a secondpolypeptide portion that binds to DNA, such as the DNA vector encodingthe individual fusion protein. When transformed host cells are culturedunder conditions that allow for expression of the fusion protein, thefusion protein binds to the DNA vector encoding it. Upon lysis of thehost cell, the fusion protein/vector DNA complexes can be screenedagainst a predetermined macromolecule in much the same way asbacteriophage particles are screened in the phage-based display system,with the replication and sequencing of the DNA vectors in the selectedfusion protein/vector DNA complexes serving as the basis foridentification of the selected library peptide sequence(s).

Other systems for generating libraries of peptides and like polymershave aspects of both the recombinant and in vitro chemical synthesismethods. In these hybrid methods, cell-free enzymatic machinery isemployed to accomplish the in vitro synthesis of the library members(i.e., peptides or polynucleotides). In one type of method, RNAmolecules with the ability to bind a predetermined protein or apredetermined dye molecule were selected by alternate rounds ofselection and PCR amplification (Tuerk and Gold (1990) Science 249: 505;Ellington and Szostak (1990) Nature 346: 818). A similar technique wasused to identify DNA sequences which bind a predetermined humantranscription factor (Thiesen and Bach (1990) Nucleic Acids Res. 18:3203; Beaudry and Joyce (1992) Science 257; 635; PCT patent publicationNos. 92/05258 and 92/14843). In a similar fashion, the technique of invitro translation has been used to synthesize proteins of interest andhas been proposed as a method for generating large libraries ofpeptides. These methods which rely upon in vitro translation, generallycomprising stabilized polysome complexes, are described further in PCTpatent publication Nos. 88/08453, 90/05785, 90/07003, 91/02076,91/05058, and 92/02536. Applicants have described methods in whichlibrary members comprise a fusion protein having a first polypeptideportion with DNA binding activity and a second polypeptide portionhaving the library member unique peptide sequence; such methods aresuitable for use in cell-free in vitro selection formats, among others.

The displayed peptide sequences can be of varying lengths, typicallyfrom 3-5000 amino acids long or longer, frequently from 5-100 aminoacids long, and often from about 8-15 amino acids long. A library cancomprise library members having varying lengths of displayed peptidesequence, or may comprise library members having a fixed length ofdisplayed peptide sequence. Portions or all of the displayed peptidesequence(s) can be random, pseudorandom, defined set kernal, fixed, orthe like. The present display methods include methods for in vitro andin vivo display of single-chain antibodies, such as nascent scFv onpolysomes or scFv displayed on phage, which enable large-scale screeningof scFv libraries having broad diversity of variable region sequencesand binding specificities.

The present invention also provides random, pseudorandom, and definedsequence framework peptide libraries and methods for generating andscreening those libraries to identify useful compounds (e.g., peptides,including single-chain antibodies) that bind to receptor molecules orepitopes of interest or gene products that modify peptides or RNA in adesired fashion. The random, pseudorandom, and defined sequenceframework peptides are produced from libraries of peptide librarymembers that comprise displayed peptides or displayed single-chainantibodies attached to a polynucleotide template from which thedisplayed peptide was synthesized. The mode of attachment may varyaccording to the specific embodiment of the invention selected, and caninclude encapsidation in a phage particle or incorporation in a cell.

A method of affinity enrichment allows a very large library of peptidesand single-chain antibodies to be screened and the polynucleotidesequence encoding the desired peptide(s) or single-chain antibodies tobe selected. The polynucleotide can then be isolated and shuffled torecombine combinatorially the amino acid sequence of the selectedpeptide(s) (or predetermined portions thereof) or single-chainantibodies (or just V_(H), V_(L), or CDR portions thereof). Using thesemethods, one can identify a peptide or single-chain antibody as having adesired binding affinity for a molecule and can exploit the process ofshuffling to converge rapidly to a desired high-affinity peptide orscFv. The peptide or antibody can then be synthesized in bulk byconventional means for any suitable use (e.g., as a therapeutic ordiagnostic agent).

A significant advantage of the present invention is that no priorinformation regarding an expected ligand structure is required toisolate peptide ligands or antibodies of interest. The peptideidentified can have biological activity, which is meant to include atleast specific binding affinity for a selected receptor molecule and, insome instances, will further include the ability to block the binding ofother compounds, to stimulate or inhibit metabolic pathways, to act as asignal or messenger, to stimulate or inhibit cellular activity, and thelike.

The present invention also provides a method for shuffling a pool ofpolynucleotide sequences selected by affinity screening a library ofpolysomes displaying nascent peptides (including single-chainantibodies) for library members which bind to a predetermined receptor(e.g., a mammalian proteinaceous receptor such as, for example, apeptidergic hormone receptor, a cell surface receptor, an intracellularprotein which binds to other protein(s) to form intracellular proteincomplexes such as heterodimers and the like) or epitope (e.g., animmobilized protein, glycoprotein, oligosaccharide, and the like).

Polynucleotide sequences selected in a first selection round (typicallyby affinity selection for binding to a receptor (e.g., a ligand) by anyof these methods are pooled and the pool(s) is/are shuffled by in vitroand/or in vivo recombination to produce a shuffled pool comprising apopulation of recombined selected polynucleotide sequences. Therecombined selected polynucleotide sequences are subjected to at leastone subsequent selection round. The polynucleotide sequences selected inthe subsequent selection round(s) can be used directly, sequenced,and/or subjected to one or more additional rounds of shuffling andsubsequent selection. Selected sequences can also be backcrossed withpolynucleotide sequences encoding neutral sequences (i.e., havinginsubstantial functional effect on binding), such as for example bybackcrossing with a wild-type or naturally-occurring sequencesubstantially identical to a selected sequence to produce native-likefunctional peptides, which may be less immunogenic. Generally, duringbackcrossing subsequent selection is applied to retain the property ofbinding to the predetermined receptor (ligand).

Prior to or concomitant with the shuffling of selected sequences, thesequences can be mutagenized. In one embodiment, selected librarymembers are cloned in a prokaryotic vector (e.g., plasmid, phagemid, orbacteriophage) wherein a collection of individual colonies (or plaques)representing discrete library members are produced. Individual selectedlibrary members can then be manipulated (e.g., by site-directedmutagenesis, cassette mutagenesis, chemical mutagenesis, PCRmutagenesis, and the like) to generate a collection of library membersrepresenting a kernal of sequence diversity based on the sequence of theselected library member. The sequence of an individual selected librarymember or pool can be manipulated to incorporate random mutation,pseudorandom mutation, defined kernal mutation (i.e., comprising variantand invariant residue positions and/or comprising variant residuepositions which can comprise a residue selected from a defined subset ofamino acid residues), codon-based mutation, and the like, eithersegmentally or over the entire length of the individual selected librarymember sequence. The mutagenized selected library members are thenshuffled by in vitro and/or in vivo recombinatorial shuffling asdisclosed herein.

The invention also provides peptide libraries comprising a plurality ofindividual library members of the invention, wherein (1) each individuallibrary member of said plurality comprises a sequence produced byshuffling of a pool of selected sequences, and (2) each individuallibrary member comprises a variable peptide segment sequence orsingle-chain antibody segment sequence which is distinct from thevariable peptide segment sequences or single-chain antibody sequences ofother individual library members in said plurality (although somelibrary members may be present in more than one copy per library due touneven amplification, stochastic probability, or the like).

The invention also provides a product-by-process, wherein selectedpolynucleotide sequences having (or encoding a peptide having) apredetermined binding specificity are formed by the process of: (1)screening a displayed peptide or displayed single-chain antibody libraryagainst a predetermined receptor (e.g., ligand) or epitope (e.g.,antigen macromolecule) and identifying and/or enriching library memberswhich bind to the predetermined receptor or epitope to produce a pool ofselected library members, (2) shuffling by recombination the selectedlibrary members (or amplified or cloned copies thereof) which binds thepredetermined epitope and has been thereby isolated and/or enriched fromthe library to generate a shuffled library, and (3) screening theshuffled library against the predetermined receptor (e.g., ligand) orepitope (e.g., antigen macromolecule) and identifying and/or enrichingshuffled library members which bind to the predetermined receptor orepitope to produce a pool of selected shuffled library members.

Antibody Display and Screening Methods

The present method can be used to shuffle, by in vitro and/or in vivorecombination by any of the disclosed methods, and in any combination,polynucleotide sequences selected by antibody display methods, whereinan associated polynucleotide encodes a displayed antibody which isscreened for a phenotype (e.g., for affinity for binding a predeterminedantigen (ligand).

Various molecular genetic approaches have been devised to capture thevast immunological repertoire represented by the extremely large numberof distinct variable regions which can be present in immunoglobulinchains. The naturally-occurring germline immunoglobulin heavy chainlocus is composed of separate tandem arrays of variable (V) segmentgenes located upstream of a tandem array of diversity (D) segment genes,which are themselves located upstream of a tandem array of joining (J)region genes, which are located upstream of the constant (C_(H)) regiongenes. During B lymphocyte development, V-D-J rearrangement occurswherein a heavy chain variable region gene (V_(H)) is formed byrearrangement to form a fused D-J segment followed by rearrangement witha V segment to form a V-D-J joined product gene which, if productivelyrearranged, encodes a functional variable region (V_(H)) of a heavychain. Similarly, light chain loci rearrange one of several V segmentswith one of several J segments to form a gene encoding the variableregion (V_(L)) of a light chain.

The vast repertoire of variable regions possible in immunoglobulinsderives in part from the numerous combinatorial possibilities of joiningV and J segments (and, in the case of heavy chain loci, D segments)during rearrangement in B cell development. Additional sequencediversity in the heavy chain variable regions arises from non-uniformrearrangements of the D segments during V-D-J joining and from N regionaddition. Further, antigen-selection of specific B cell clones selectsfor higher affinity variants having nongermline mutations in one or bothof the heavy and light chain variable regions; a phenomenon referred toas "affinity maturation" or "affinity sharpening". Typically, these"affinity sharpening" mutations cluster in specific areas of thevariable region, most commonly in the complementarity-determiningregions (CDRs).

In order to overcome many of the limitations in producing andidentifying high-affinity immunoglobulins through antigen-stimulated Bcell development (i.e., immunization), various prokaryotic expressionsystems have been developed that can be manipulated to producecombinatorial antibody libraries which may be screened for high-affinityantibodies to specific antigens. Recent advances in the expression ofantibodies in Escherichia coli and bacteriophage systems (see,"Alternative Peptide Display Methods", infra) have raised thepossibility that virtually any specificity can be obtained by eithercloning antibody genes from characterized hybridomas or by de novoselection using antibody gene libraries (e.g., from Ig cDNA).

Combinatorial libraries of antibodies have been generated inbacteriophage lambda expression systems which may be screened asbacteriophage plaques or as colonies of lysogens (Huse et al. (1989)Science 246: 1275; Caton and Koprowski (1990) Proc. Natl. Acad. Sci.(U.S.A.) 87: 6450; Mullinax et al (1990) Proc. Natl. Acad. Sci. (U.S.A.)87: 8095; Persson et al. (1991) Proc. Natl. Acad. Sci. (U.S.A.) 88:2432). Various embodiments of bacteriophage antibody display librariesand lambda phage expression libraries have been described (Kang et al.(1991) Proc. Natl. Acad. Sci. (U.S.A.) 88: 4363; Clackson et al. (1991)Nature 352: 624; McCafferty et al. (1990) Nature 348: 552; Burton et al.(1991) Proc. Natl. Acad. Sci. (U.S.A.) 88: 10134; Hoogenboom et al.(1991) Nucleic Acids Res. 19: 4133; Chang et al. (1991) J. Immunol. 147:3610; Breitling et al. (1991) Gene 104: 147; Marks et al. (1991) J. Mol.Biol. 222: 581; Barbas et al. (1992) Proc. Natl. Acad. Sci. (U.S.A.) 89:4457; Hawkins and Winter (1992) J. Immunol. 22: 867; Marks et al. (1992)Biotechnology 10: 779; Marks et al. (1992) J. Biol. Chem. 267: 16007;Lowman et al (1991) Biochemistry 30: 10832; Lerner et al. (1992) Science258: 1313, incorporated herein by reference). Typically, a bacteriophageantibody display library is screened with a receptor (e.g., polypeptide,carbohydrate, glycoprotein, nucleic acid) that is immobilized (e.g., bycovalent linkage to a chromatography resin to enrich for reactive phageby affinity chromatography) and/or labeled (e.g., to screen plaque orcolony lifts).

One particularly advantageous approach has been the use of so-calledsingle-chain fragment variable (scFv) libraries (Marks et al. (1992)Biotechnology 10: 779; Winter G and Milstein C (1991) Nature 349: 293;Clackson et al. (1991) op.cit.; Marks et al. (1991) J. Mol. Biol. 222:581; Chaudhary et al. (1990) Proc. Natl. Acad. Sci. (USA) 87: 1066;Chiswell et al. (1992) TIBTECH 10: 80; McCafferty et al. (1990) op.cit.;and Huston et al. (1988) Proc. Natl. Acad. Sci. (USA) 85: 5879). Variousembodiments of scFv libraries displayed on bacteriophage coat proteinshave been described.

Beginning in 1988, single-chain analogues of Fv fragments and theirfusion proteins have been reliably generated by antibody engineeringmethods. The first step generally involves obtaining the genes encodingV_(H) and V_(L) domains with desired binding properties; these V genesmay be isolated from a specific hybridoma cell line, selected from acombinatorial V-gene library, or made by V gene synthesis. Thesingle-chain Fv is formed by connecting the component V genes with anoligonucleotide that encodes an appropriately designed linker peptide,such as (Gly-Gly-Gly-Gly-Ser)₃ or equivalent linker peptide(s). Thelinker bridges the C-terminus of the first V region and N-terminus ofthe second, ordered as either V_(H) -linker-V_(L) or V_(L)-linker-V_(H). In principle, the scFv binding site can faithfullyreplicate both the affinity and specificity of its parent antibodycombining site.

Thus, scFv fragments are comprised of V_(H) and V_(L) domains linkedinto a single polypeptide chain by a flexible linker peptide. After thescFv genes are assembled, they are cloned into a phagemid and expressedat the tip of the M13 phage (or similar filamentous bacteriophage) asfusion proteins with the bacteriophage pIII (gene 3) coat protein.Enriching for phage expressing an antibody of interest is accomplishedby panning the recombinant phage displaying a population scFv forbinding to a predetermined epitope (e.g., target antigen, receptor).

The linked polynucleotide of a library member provides the basis forreplication of the library member after a screening or selectionprocedure, and also provides the basis for the determination, bynucleotide sequencing, of the identity of the displayed peptide sequenceor V_(H) and V_(L) amino acid sequence. The displayed peptide(s) orsingle-chain antibody (e.g., scFv) and/or its V_(H) and V_(L) domains ortheir CDRs can be cloned and expressed in a suitable expression system.Often polynucleotides encoding the isolated V_(H) and V_(L) domains willbe ligated to polynucleotides encoding constant regions (C_(H) andC_(L)) to form polynucleotides encoding complete antibodies (e.g.,chimeric or fully-human), antibody fragments, and the like. Oftenpolynucleotides encoding the isolated CDRs will be grafted intopolynucleotides encoding a suitable variable region framework (andoptionally constant regions) to form polynucleotides encoding completeantibodies (e.g., humanized or fully-human), antibody fragments, and thelike. Antibodies can be used to isolate preparative quantities of theantigen by immunoaffinity chromatography. Various other uses of suchantibodies are to diagnose and/or stage disease (e.g., neoplasia), andfor therapeutic application to treat disease, such as for example:neoplasia, autoimmune disease, AIDS, cardiovascular disease, infections,and the like.

Various methods have been reported for increasing the combinatorialdiversity of a scFv library to broaden the repertoire of binding species(idiotype spectrum). The use of PCR has permitted the variable regionsto be rapidly cloned either from a specific hybridoma source or as agene library from non-immunized cells, affording combinatorial diversityin the assortment of V_(H) and V_(L) cassettes which can be combined.Furthermore, the V_(H) and V_(L) cassettes can themselves bediversified, such as by random, pseudorandom, or directed mutagenesis.Typically, V_(H) and V_(L) cassettes are diversified in or near thecomplementarity-determining regions (CDRs), often the third CDR, CDR3.Enzymatic inverse PCR mutagenesis has been shown to be a simple andreliable method for constructing relatively large libraries of scFvsite-directed mutants (Stemmer et al. (1993) Biotechniques 14: 256), ashas error-prone PCR and chemical mutagenesis (Deng et al. (1994) J.Biol. Chem. 269: 9533). Riechmann et al. (1993) Biochemistry 32: 8848showed semirational design of an antibody scFv fragment usingsite-directed randomization by degenerate oligonucleotide PCR andsubsequent phage display of the resultant scFv mutants. Barbas et al.(1992) op.cit. attempted to circumvent the problem of limited repertoiresizes resulting from using biased variable region sequences byrandomizing the sequence in a synthetic CDR region of a human tetanustoxoid-binding Fab.

CDR randomization has the potential to create approximately 1×10²⁰ CDRsfor the heavy chain CDR3 alone, and a roughly similar number of variantsof the heavy chain CDR1 and CDR2, and light chain CDR1-3 variants. Takenindividually or together, the combinatorics of CDR randomization ofheavy and/or light chains requires generating a prohibitive number ofbacteriophage clones to produce a clone library representing allpossible combinations, the vast majority of which will be non-binding.Generation of such large numbers of primary transformants is notfeasible with current transformation technology and bacteriophagedisplay systems. For example, Barbas et al. (1992) op.cit. onlygenerated 5×10⁷ transformants, which represents only a tiny fraction ofthe potential diversity of a library of thoroughly randomized CDRs.

Despite these substantial limitations, bacteriophage display of scFvhave already yielded a variety of useful antibodies and antibody fusionproteins. A bispecific single chain antibody has been shown to mediateefficient tumor cell lysis (Gruber et al. (1994) J. Immunol. 152: 5368).Intracellular expression of an anti-Rev scFv has been shown to inhibitHIV-1 virus replication in vitro (Duan et al. (1994) Proc. Natl. Acad.Sci. (USA) 91: 5075), and intracellular expression of an anti-p21^(ras)scFv has been shown to inhibit meiotic maturation of Xenopus oocytes(Biocca et al. (1993) Biochem. Biophys. Res. Commun. 197: 422.Recombinant scFv which can be used to diagnose HIV infection have alsobeen reported, demonstrating the diagnostic utility of scFv (Lilley etal. (1994) J. Immunol. Meth. 171: 211). Fusion proteins wherein an scFvis linked to a second polypeptide, such as a toxin or fibrinolyticactivator protein, have also been reported (Holvost et al. (1992) Eur.J. Biochem. 210: 945; Nicholls et al. (1993) J. Biol. Chem. 268: 5302).

If it were possible to generate scFv libraries having broader antibodydiversity and overcoming many of the limitations of conventional CDRmutagenesis and randomization methods which can cover only a very tinyfraction of the potential sequence combinations, the number and qualityof scFv antibodies suitable for therapeutic and diagnostic use could bevastly improved. To address this, the in vitro and in vivo shufflingmethods of the invention are used to recombine CDRs which have beenobtained (typically via PCR amplification or cloning) from nucleic acidsobtained from selected displayed antibodies. Such displayed antibodiescan be displayed on cells, on bacteriophage particles, on polysomes, orany suitable antibody display system wherein the antibody is associatedwith its encoding nucleic acid(s). In a variation, the CDRs areinitially obtained from mRNA (or cDNA) from antibody-producing cells(e.g., plasma cells/splenocytes from an immunized wild-type mouse, ahuman, or a transgenic mouse capable of making a human antibody as inWO92/03918, WO93/12227, and WO94/25585), including hybridomas derivedtherefrom.

Polynucleotide sequences selected in a first selection round (typicallyby affinity selection for displayed antibody binding to an antigen(e.g., a ligand) by any of these methods are pooled and the pool(s)is/are shuffled by in vitro and/or in vivo recombination, especiallyshuffling of CDRs (typically shuffling heavy chain CDRs with other heavychain CDRs and light chain CDRs with other light chain CDRs) to producea shuffled pool comprising a population of recombined selectedpolynucleotide sequences. The recombined selected polynucleotidesequences are expressed in a selection format as a displayed antibodyand subjected to at least one subsequent selection round. Thepolynucleotide sequences selected in the subsequent selection round(s)can be used directly, sequenced, and/or subjected to one or moreadditional rounds of shuffling and subsequent selection until anantibody of the desired binding affinity is obtained. Selected sequencescan also be backcrossed with polynucleotide sequences encoding neutralantibody framework sequences (i.e., having insubstantial functionaleffect on antigen binding), such as for example by backcrossing with ahuman variable region framework to produce human-like sequenceantibodies. Generally, during backcrossing subsequent selection isapplied to retain the property of binding to the predetermined antigen.

Alternatively, or in combination with the noted variations, the valencyof the target epitope may be varied to control the average bindingaffinity of selected scFv library members. The target epitope can bebound to a surface or substrate at varying densities, such as byincluding a competitor epitope, by dilution, or by other method known tothose in the art. A high density (valency) of predetermined epitope canbe used to enrich for scFv library members which have relatively lowaffinity, whereas a low density (valency) can preferentially enrich forhigher affinity scFv library members.

For generating diverse variable segments, a collection of syntheticoligonucleotides encoding random, pseudorandom, or a defined sequencekernal set of peptide sequences can be inserted by ligation into apredetermined site (e.g., a CDR). Similarly, the sequence diversity ofone or more CDRs of the single-chain antibody cassette(s) can beexpanded by mutating the CDR(s) with site-directed mutagenesis,CDR-replacement, and the like. The resultant DNA molecules can bepropagated in a host for cloning and amplification prior to shuffling,or can be used directly (i.e., may avoid loss of diversity which mayoccur upon propagation in a host cell) and the selected library memberssubsequently shuffled.

Displayed peptide/polynucleotide complexes (library members) whichencode a variable segment peptide sequence of interest or a single-chainantibody of interest are selected from the library by an affinityenrichment technique. This is accomplished by means of a immobilizedmacromolecule or epitope specific for the peptide sequence of interest,such as a receptor, other macromolecule, or other epitope species.Repeating the affinity selection procedure provides an enrichment oflibrary members encoding the desired sequences, which may then beisolated for pooling and shuffling, for sequencing, and/or for furtherpropagation and affinity enrichment.

The library members without the desired specificity are removed bywashing. The degree and stringency of washing required will bedetermined for each peptide sequence or single-chain antibody ofinterest and the immobilized predetermined macromolecule or epitope. Acertain degree of control can be exerted over the bindingcharacteristics of the nascent peptide/DNA complexes recovered byadjusting the conditions of the binding incubation and the subsequentwashing. The temperature, pH, ionic strength, divalent cationsconcentration, and the volume and duration of the washing will selectfor nascent peptide/DNA complexes within particular ranges of affinityfor the immobilized macromolecule. Selection based on slow dissociationrate, which is usually predictive of high affinity, is often the mostpractical route. This may be done either by continued incubation in thepresence of a saturating amount of free predetermined macromolecule, orby increasing the volume, number, and length of the washes. In eachcase, the rebinding of dissociated nascent peptide/DNA or peptide/RNAcomplex is prevented, and with increasing time, nascent peptide/DNA orpeptide/RNA complexes of higher and higher affinity are recovered.

Additional modifications of the binding and washing procedures may beapplied to find peptides with special characteristics. The affinities ofsome peptides are dependent on ionic strength or cation concentration.This is a useful characteristic for peptides that will be used inaffinity purification of various proteins when gentle conditions forremoving the protein from the peptides are required.

One variation involves the use of multiple binding targets (multipleepitope species, multiple receptor species), such that a scFv librarycan be simultaneously screened for a multiplicity of scFv which havedifferent binding specificities. Given that the size of a scFv libraryoften limits the diversity of potential scFv sequences, it is typicallydesirable to us scFv libraries of as large a size as possible. The timeand economic considerations of generating a number of very largepolysome scFv-display libraries can become prohibitive. To avoid thissubstantial problem, multiple predetermined epitope species (receptorspecies) can be concomitantly screened in a single library, orsequential screening against a number of epitope species can be used. Inone variation, multiple target epitope species, each encoded on aseparate bead (or subset of beads), can be mixed and incubated with apolysome-display scFv library under suitable binding conditions. Thecollection of beads, comprising multiple epitope species, can then beused to isolate, by affinity selection, scFv library members. Generally,subsequent affinity screening rounds can include the same mixture ofbeads, subsets thereof, or beads containing only one or two individualepitope species. This approach affords efficient screening, and iscompatible with laboratory automation, batch processing, and highthroughput screening methods.

A variety of techniques can be used in the present invention todiversify a peptide library or single-chain antibody library, or todiversify, prior to or concomitant with shuffling, around variablesegment peptides or V_(H), V_(L), or CDRs found in early rounds ofpanning to have sufficient binding activity to the predeterminedmacromolecule or epitope. In one approach, the positive selectedpeptide/polynucleotide complexes (those identified in an early round ofaffinity enrichment) are sequenced to determine the identity of theactive peptides. Oligonucleotides are then synthesized based on theseactive peptide sequences, employing a low level of all basesincorporated at each step to produce slight variations of the primaryoligonucleotide sequences. This mixture of (slightly) degenerateoligonucleotides is then cloned into the variable segment sequences atthe appropriate locations. This method produces systematic, controlledvariations of the starting peptide sequences, which can then beshuffled. It requires, however, that individual positive nascentpeptide/polynucleotide complexes be sequenced before mutagenesis, andthus is useful for expanding the diversity of small numbers of recoveredcomplexes and selecting variants having higher binding affinity and/orhigher binding specificity. In a variation, mutagenic PCR amplificationof positive selected peptide/polynucleotide complexes (especially of thevariable region sequences, the amplification products of which areshuffled in vitro and/or in vivo and one or more additional rounds ofscreening is done prior to sequencing. The same general approach can beemployed with single-chain antibodies in order to expand the diversityand enhance the binding affinity/specificity, typically by diversifyingCDRs or adjacent framework regions prior to or concomitant withshuffling. If desired, shuffling reactions can be spiked with mutagenicoligonucleotides capable of in vitro recombination with the selectedlibrary members can be included. Thus, mixtures of syntheticoligonucleotides and PCR fragments (synthesized by error-prone orhigh-fidelity methods) can be added to the in vitro shuffling mix and beincorporated into resulting shuffled library members (shufflants).

The present invention of shuffling enables the generation of a vastlibrary of CDR-variant single-chain antibodies. One way to generate suchantibodies is to insert synthetic CDRs into the single-chain antibodyand/or CDR randomization prior to or concomitant with shuffling. Thesequences of the synthetic CDR cassettes are selected by referring toknown sequence data of human CDR and are selected in the discretion ofthe practitioner according to the following guidelines: synthetic CDRswill have at least 40 percent positional sequence identity to known CDRsequences, and preferably will have at least 50 to 70 percent positionalsequence identity to known CDR sequences. For example, a collection ofsynthetic CDR sequences can be generated by synthesizing a collection ofoligonucleotide sequences on the basis of naturally-occurring human CDRsequences listed in Kabat et al. (1991) op.cit.; the pool(s) ofsynthetic CDR sequences are calculated to encode CDR peptide sequenceshaving at least 40 percent sequence identity to at least one knownnaturally-occurring human CDR sequence. Alternatively, a collection ofnaturally-occurring CDR sequences may be compared to generate consensussequences so that amino acids used at a residue position frequently(i.e., in at least 5 percent of known CDR sequences) are incorporatedinto the synthetic CDRs at the corresponding position(s). Typically,several (e.g., 3 to about 50) known CDR sequences are compared andobserved natural sequence variations between the known CDRs aretabulated, and a collection of oligonucleotides encoding CDR peptidesequences encompassing all or most permutations of the observed naturalsequence variations is synthesized. For example but not for limitation,if a collection of human V_(H) CDR sequences have carboxy-terminal aminoacids which are either Tyr, Val, Phe, or Asp, then the pool(s) ofsynthetic CDR oligonucleotide sequences are designed to allow thecarboxy-terminal CDR residue to be any of these amino acids. In someembodiments, residues other than those which naturally-occur at aresidue position in the collection of CDR sequences are incorporated:conservative amino acid substitutions are frequently incorporated and upto 5 residue positions may be varied to incorporate non-conservativeamino acid substitutions as compared to known naturally-occurring CDRsequences. Such CDR sequences can be used in primary library members(prior to first round screening) and/or can be used to spike in vitroshuffling reactions of selected library member sequences. Constructionof such pools of defined and/or degenerate sequences will be readilyaccomplished by those of ordinary skill in the art.

The collection of synthetic CDR sequences comprises at least one memberthat is not known to be a naturally-occurring CDR sequence. It is withinthe discretion of the practitioner to include or not include a portionof random or pseudorandom sequence corresponding to N region addition inthe heavy chain CDR; the N region sequence ranges from 1 nucleotide toabout 4 nucleotides occurring at V-D and D-J junctions. A collection ofsynthetic heavy chain CDR sequences comprises at least about 100 uniqueCDR sequences, typically at least about 1,000 unique CDR sequences,preferably at least about 10,000 unique CDR sequences, frequently morethan 50,000 unique CDR sequences; however, usually not more than about1×10⁶ unique CDR sequences are included in the collection, althoughoccasionally 1×10⁷ to 1×10⁸ unique CDR sequences are present, especiallyif conservative amino acid substitutions are permitted at positionswhere the conservative amino acid substituent is not present or is rare(i.e., less than 0.1 percent) in that position in naturally-occurringhuman CDRs. In general, the number of unique CDR sequences included in alibrary should not exceed the expected number of primary transformantsin the library by more than a factor of 10. Such single-chain antibodiesgenerally bind to a predetermined antigen (e.g., the immunogen) with anaffinity of about at least 1×10⁷ M⁻¹, preferably with an affinity ofabout at least 5×10⁷ M⁻¹, more preferably with an affinity of at least1×10⁸ M⁻¹ to 1×10⁹ M⁻¹ or more, sometimes up to 1×10¹ M⁻¹ or more.Frequently, the predetermined antigen is a human protein, such as forexample a human cell surface antigen (e.g., CD4, CD8, IL-2 receptor, EGFreceptor, PDGF receptor), other human biological macromolecule (e.g.,thrombomodulin, protein C, carbohydrate antigen, sialyl Lewis antigen,L-selectin), or nonhuman disease associated macromolecule (e.g.,bacterial LPS, virion capsid protein or envelope glycoprotein) and thelike.

High affinity single-chain antibodies of the desired specificity can beengineered and expressed in a variety of systems. For example, scFv havebeen produced in plants (Firek et al. (1993) Plant Mol. Biol. 23: 861)and can be readily made in prokaryotic systems (Owens, R. J. and Young,R. J. (1994) J. Immunol. Meth. 168: 149; Johnson, S. and Bird, R. E.(1991) Methods Enzymol. 203: 88). Furthermore, the single-chainantibodies can be used as a basis for constructing whole antibodies orvarious fragments thereof (Kettleborough et al. (1994) Eur. J. Immunol.24: 952). The variable region encoding sequence may be isolated (e.g.,by PCR amplification or subcloning) and spliced to a sequence encoding adesired human constant region to encode a human sequence antibody moresuitable for human therapeutic uses where immunogenicity is preferablyminimized. The polynucleotide(s) having the resultant fully humanencoding sequence(s) can be expressed in a host cell (e.g., from anexpression vector in a mammalian cell) and purified for pharmaceuticalformulation.

The DNA expression constructs will typically include an expressioncontrol DNA sequence operably linked to the coding sequences, includingnaturally-associated or heterologous promoter regions. Preferably, theexpression control sequences will be eukaryotic promoter systems invectors capable of transforming or transfecting eukaryotic host cells.Once the vector has been incorporated into the appropriate host, thehost is maintained under conditions suitable for high level expressionof the nucleotide sequences, and the collection and purification of themutant "engineered" antibodies.

As stated previously, the DNA sequences will be expressed in hosts afterthe sequences have been operably linked to an expression controlsequence (i.e., positioned to ensure the transcription and translationof the structural gene). These expression vectors are typicallyreplicable in the host organisms either as episomes or as an integralpart of the host chromosomal DNA. Commonly, expression vectors willcontain selection markers, e.g., tetracycline or neomycin, to permitdetection of those cells transformed with the desired DNA sequences(see, e.g., U.S. Pat. No. 4,704,362, which is incorporated herein byreference).

In addition to eukaryotic microorganisms such as yeast, mammalian tissuecell culture may also be used to produce the polypeptides of the presentinvention (see, Winnacker, "From Genes to Clones," VCH Publishers, N.Y.,N.Y. (1987), which is incorporated herein by reference). Eukaryoticcells are actually preferred, because a number of suitable host celllines capable of secreting intact immunoglobulins have been developed inthe art, and include the CHO cell lines, various COS cell lines, HeLacells, myeloma cell lines, etc, but preferably transformed B-cells orhybridomas. Expression vectors for these cells can include expressioncontrol sequences, such as an origin of replication, a promoter, anenhancer (Queen et al. (1986) Immunol. Rev. 89: 49), and necessaryprocessing information sites, such as ribosome binding sites, RNA splicesites, polyadenylation sites, and transcriptional terminator sequences.Preferred expression control sequences are promoters derived fromimmunoglobulin genes, cytomegalovirus, SV40, Adenovirus, BovinePapilloma Virus, and the like.

Eukaryotic DNA transcription can be increased by inserting an enhancersequence into the vector. Enhancers are cis-acting sequences of between10 to 300 bp that increase transcription by a promoter. Enhancers caneffectively increase transcription when either 5' or 3' to thetranscription unit. They are also effective if located within an intronor within the coding sequence itself. Typically, viral enhancers areused, including SV40 enhancers, cytomegalovirus enhancers, polyomaenhancers, and adenovirus enhancers. Enhancer sequences from mammaliansystems are also commonly used, such as the mouse immunoglobulin heavychain enhancer.

Mammalian expression vector systems will also typically include aselectable marker gene. Examples of suitable markers include, thedihydrofolate reductase gene (DHFR), the thymidine kinase gene (TK), orprokaryotic genes conferring drug resistance. The first two marker genesprefer the use of mutant cell lines that lack the ability to growwithout the addition of thymidine to the growth medium. Transformedcells can then be identified by their ability to grow onnon-supplemented media. Examples of prokaryotic drug resistance genesuseful as markers include genes conferring resistance to G418,mycophenolic acid and hygromycin.

The vectors containing the DNA segments of interest can be transferredinto the host cell by well-known methods, depending on the type ofcellular host. For example, calcium chloride transfection is commonlyutilized for prokaryotic cells, whereas calcium phosphate treatment.lipofection, or electroporation may be used for other cellular hosts.Other methods used to transform mammalian cells include the use ofPolybrene, protoplast fusion, liposomes, electroporation, andmicroinjection (see, generally, Sambrook et al., supra).

Once expressed, the antibodies, individual mutated immunoglobulinchains, mutated antibody fragments, and other immunoglobulinpolypeptides of the invention can be purified according to standardprocedures of the art, including ammonium sulfate precipitation,fraction column chromatography, gel electrophoresis and the like (see,generally, Scopes, R., Protein Purification, Springer-Verlag, N.Y.(1982)). Once purified, partially or to homogeneity as desired, thepolypeptides may then be used therapeutically or in developing andperforming assay procedures, immunofluorescent stainings, and the like(see, generally, Immunological Methods, Vols. I and II, Eds. Lefkovitsand Pernis, Academic Press, New York, N.Y. (1979 and 1981)).

The antibodies generated by the method of the present invention can beused for diagnosis and therapy. By way of illustration and notlimitation, they can be used to treat cancer, autoimmune diseases, orviral infections. For treatment of cancer, the antibodies will typicallybind to an antigen expressed preferentially on cancer cells, such aserbB-2, CEA, CD33, and many other antigens and binding members wellknown to those skilled in the art.

Yeast Two-Hybrid Screening Assays

Shuffling can also be used to recombinatorially diversify a pool ofselected library members obtained by screening a two-hybrid screeningsystem to identify library members which bind a predeterminedpolypeptide sequence. The selected library members are pooled andshuffled by in vitro and/or in vivo recombination. The shuffled pool canthen be screened in a yeast two hybrid system to select library memberswhich bind said predetermined polypeptide sequence (e.g., and SH2domain) or which bind an alternate predetermined polypeptide sequence(e.g., an SH2 domain from another protein species).

An approach to identifying polypeptide sequences which bind to apredetermined polypeptide sequence has been to use a so-called"two-hybrid" system wherein the predetermined polypeptide sequence ispresent in a fusion protein (Chien et al. (1991) Proc. Natl. Acad. Sci.(USA) 88: 9578). This approach identifies protein-protein interactionsin vivo through reconstitution of a transcriptional activator (Fields,S. and Song, O. (1989) Nature 340: 245), the yeast Gal4 transcriptionprotein. Typically, the method is based on the properties of the yeastGal4 protein, which consists of separable domains responsible forDNA-binding and transcriptional activation. Polynucleotides encoding twohybrid proteins, one consisting of the yeast Gal4 DNA-binding domainfused to a polypeptide sequence of a known protein and the otherconsisting of the Gal4 activation domain fused to a polypeptide sequenceof a second protein, are constructed and introduced into a yeast hostcell. Intermolecular binding between the two fusion proteinsreconstitutes the Gal4 DNA-binding domain with the Gal4 activationdomain, which leads to the transcriptional activation of a reporter gene(e.g., lacZ, HIS3) which is operably linked to a Gal4 binding site.Typically, the two-hybrid method is used to identify novel polypeptidesequences which interact with a known protein (Silver, S. C. and Hunt,S. W. (1993) Mol. Biol. Rep. 17: 155; Durfee et al. (1993) Genes Devel.7; 555; Yang et al. (1992) Science 257: 680; Luban et al. (1993) Cell73: 1067; Hardy et al. (1992) Genes Devel. 6; 801; Bartel et al. (1993)Biotechniques 14: 920; and Vojtek et al. (1993) Cell 74: 205). However,variations of the two-hybrid method have been used to identify mutationsof a known protein that affect its binding to a second known protein(Li, B. and Fields, S. (1993) FASEB J. 7: 957; Lalo et al. (1993) Proc.Natl. Acad. Sci. (USA) 90: 5524; Jackson et al. (1993) Mol. Cell. Biol.13; 2899; and Madura et al. (1993) J. Biol. Chem. 268: 12046).Two-hybrid systems have also been used to identify interactingstructural domains of two known proteins (Bardwell et al. (1993) med.Microbiol. 8: 1177; Chakraborty et al. (1992) J. Biol. Chem. 267: 17498;Staudinger et al. (1993) J. Biol. Chem. 268: 4608; and Milne, G. T. andWeaver, D. T. (1993) Genes Devel. 7; 1755) or domains responsible foroligomerization of a single protein (Iwabuchi et al. (1993) Oncogene 8;1693; Bogerd et al. (1993) J. Virol. 67: 5030). Variations of two-hybridsystems have been used to study the in vivo activity of a proteolyticenzyme (Dasmahapatra et al. (1992) Proc. Natl. Acad. Sci. (USA) 89:4159). Alternatively, an E. coli/BCCP interactive screening system(Germino et al. (1993) Proc. Natl. Acad. Sci. (U.S.A.) 90: 933;Guarente, L. (1993) Proc. Natl. Acad. Sci. (U.S.A.) 90: 1639) can beused to identify interacting protein sequences (i.e., protein sequenceswhich heterodimerize or form higher order heteromultimers). Sequencesselected by a two-hybrid system can be pooled and shuffled andintroduced into a two-hybrid system for one or more subsequent rounds ofscreening to identify polypeptide sequences which bind to the hybridcontaining the predetermined binding sequence. The sequences thusidentified can be compared to identify consensus sequence(s) andconsensus sequence kernals.

As can be appreciated from the disclosure above, the present inventionhas a wide variety of applications. Accordingly, the following examplesare offered by way of illustration, not by way of limitation.

In the examples below, the following abbreviations have the followingmeanings. If not defined below, then the abbreviations have their artrecognized meanings.

    ______________________________________                                        ml =        milliliter                                                        μl =     microliters                                                       μM =     micromolar                                                        nM =        nanomolar                                                         PBS =       phosphate buffered saline                                         ng =        nanograms                                                         μg =     micrograms                                                        IPTG =      isopropylthio-β-D-galactoside                                bp =        basepairs                                                         kb =        kilobasepairs                                                     dNTP =      deoxynucleoside triphosphates                                     PCR =       polymerase chain reaction                                         X-gal =     5-bromo-4-chloro-3-indolyl-β-D-galactoside                   DNAseI =    deoxyribonuclease                                                 PBS =       phosphate buffered saline                                         CDR =       complementarity determining regions                               MIC =       minimum inhibitory concentration                                  scFv =      single-chain Fv fragment of an antibody                           ______________________________________                                    

In general, standard techniques of recombination DNA technology aredescribed in various publications, e.g. Sambrook et al., 1989, MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratory; Ausubel etal., 1987, Current Protocols in Molecular Biology, vols. 1 and 2 andsupplements, and Berger and Kimmel, Methods in Enzymology, Volume 152,Guide to Molecular Cloning Techniques (1987), Academic Press, Inc., SanDiego, Calif., each of which is incorporated herein in their entirety byreference. Restriction enzymes and polynucleotide modifying enzymes wereused according to the manufacturers recommendations. Oligonucleotideswere synthesized on an Applied Biosystems Inc. Model 394 DNA synthesizerusing ABI chemicals. If desired, PCR amplimers for amplifying apredetermined DNA sequence may be selected at the discretion of thepractitioner.

EXAMPLES Example 1 LacZ Alpha Gene Reassembly

1) Substrate preparation

The substrate for the reassembly reaction was the dsDNA polymerase chainreaction ("PCR") product of the wild-type LacZ alpha gene from pUC18.(FIG. 2) (28; Gene Bank No. XO2514) The primer sequences were5'AAAGCGTCGATTTTTGTGAT3' (SEQ ID NO:1) and 5'ATGGGGTTCCGCGCACATTT3' (SEQID NO:2). The free primers were removed from the PCR product by WizardPCR prep (Promega, Madison, Wis.) according to the manufacturer'sdirections. The removal of the free primers was found to be important.

2) DNAseI digestion

About 5 μg of the DNA substrate was digested with 0.15 units of DNAseI(Sigma, St. Louis, Mo.) in 100 μl of 50 mM Tris-HCl pH 7.4, 1 mM MgCl₂!, for 10-20 minutes at room temperature. The digested DNA was run on a2% low melting point agarose gel. Fragments of 10-70 basepairs (bp) werepurified from the 2% low melting point agarose gels by electrophoresisonto DE81 ion exchange paper (Whatman, Hillsborough, Oreg.). The DNAfragments were eluted from the paper with 1M NaCl and ethanolprecipitated.

3) DNA Reassembly

The purified fragments were resuspended at a concentration of 10-30ng/μl in PCR Mix (0.2 mM each dNTP, 2.2 mM MgCl₂, 50 mM KCl, 10 mMTris-HCl pH 9.0, 0.1% Triton X-100, 0.3 μl Taq DNA polymerase, 50 μltotal volume). No primers were added at this point. A reassembly programof 94° C. for 60 seconds, 30-45 cycles of 94° C. for 30 seconds, 50°-55°C. for 30 seconds, 72° C. for 30 seconds! and 5 minutes at 72° C. wasused in an MJ Research (Watertown, Mass.) PTC-150 thermocycler. The PCRreassembly of small fragments into larger sequences was followed bytaking samples of the reaction after 25, 30, 35 ,40 and 45 cycles ofreassembly (FIG. 2).

Whereas the reassembly of 100-200 bp fragments can yield a single PCRproduct of the correct size, 10-50 base fragments typically yield someproduct of the correct size, as well as products of heterogeneousmolecular weights. Most of this size heterogeneity appears to be due tosingle-stranded sequences at the ends of the products, since afterrestriction enzyme digestion a single band of the correct size isobtained.

4) PCR with primers

After dilution of the reassembly product into the PCR Mix with 0.8 μM ofeach of the above primers (SEQ ID Nos: 1 and 2) and about 15 cycles ofPCR, each cycle consisting of 94° C. for 30 seconds, 50° C. for 30seconds and 72° C. for 30 seconds!, a single product of the correct sizewas obtained (FIG. 2).

5) Cloning and analysis

The PCR product from step 4 above was digested with the terminalrestriction enzymes BamHI and EcoO109 and gel purified as describedabove in step 2. The reassembled fragments were ligated into pUC18digested with BamHI and EcoO109. E. coli were transformed with theligation mixture under standard conditions as recommended by themanufacturer (Stratagene, San Diego, Calif.) and plated on agar plateshaving 100 μg/ml ampicillin, 0.004% X-gal and 2 mM IPTG. The resultingcolonies having the HinDIII-NheI fragment which is diagnostic for the ++recombinant were identified because they appeared blue.

This Example illustrates that a 1.0 kb sequence carrying the LacZ alphagene can be digested into 10-70 bp fragments, and that these gelpurified 10-70 bp fragments can be reassembled to a single product ofthe correct size, such that 84% (N=377) of the resulting colonies areLacZ⁺ (versus 94% without shuffling; FIG. 2).

The DNA encoding the LacZ gene from the resulting LacZ⁻ colonies wassequenced with a sequencing kit (United States Biochemical Co.,Cleveland, Ohio) according to the manufacturer's instructions and thegenes were found to have point mutations due to the reassembly process(Table 1). 11/12 types of substitutions were found, and no frameshifts.

                  TABLE 1                                                         ______________________________________                                        Mutations introduced by mutagenic shuffling                                   Transitions                                                                             Frequency   Transversions                                                                            Frequency                                    ______________________________________                                        G-A       6           A-T        1                                            A-G       4           A-C        2                                            C-T       7           C-A        1                                            T-C       3           C-G        0                                                                  G-C        3                                                                  G-T        2                                                                  T-A        1                                                                  T-G        2                                            ______________________________________                                         A total of 4,437 bases of shuffled lacZ DNA were sequenced.              

The rate of point mutagenesis during DNA reassembly from 10-70 bp pieceswas determined from DNA sequencing to be 0.7% (N=4,473), which issimilar to error-prone PCR. Without being limited to any theory it isbelieved that the rate of point mutagenesis may be lower if largerfragments are used for the reassembly, or if a proofreading polymeraseis added.

When plasmid DNA from 14 of these point-mutated LacZ⁻ colonies werecombined and again reassembled/shuffled by the method described above,34% (N=291) of the resulting colonies were LacZ⁺, and these coloniespresumably arose by recombination of the DNA from different colonies.

The expected rate of reversal of a single point mutation by error-pronePCR, assuming a mutagenesis rate of 0.7% (10), would be expected to be<1%.

Thus large DNA sequences can be reassembled from a random mixture ofsmall fragments by a reaction that is surprisingly efficient and simple.One application of this technique is the recombination or shuffling ofrelated sequences based on homology.

Example 2 LacZ Gene and Whole Plasmid DNA Shuffling

1) LacZ gene shuffling

Crossover between two markers separated by 75 bases was measured usingtwo LacZ gene constructs. Stop codons were inserted in two separateareas of the LacZ alpha gene to serve as negative markers. Each markeris a 25 bp non-homologous sequence with four stop codons, of which twoare in the LacZ gene reading frame. The 25 bp non-homologous sequence isindicated in FIG. 3 by a large box. The stop codons are either boxed orunderlined. A 1:1 mixture of the two 1.0 kb LacZ templates containingthe +- and -+ versions of the LacZ alpha gene (FIG. 3) was digested withDNAseI and 100-200 bp fragments were purified as described in Example 1.The shuffling program was conducted under conditions similar to thosedescribed for reassembly in Example 1 except 0.5 μl of polymerase wasadded and the total volume was 100 μl.

After cloning, the number of blue colonies obtained was 24%; (N=386)which is close to the theoretical maximum number of blue colonies (i.e.25%), indicating that recombination between the two markers wascomplete. All of the 10 blue colonies contained the expectedHindIII-NheI restriction fragment.

2) Whole plasmid DNA shuffling

Whole 2.7 kb plasmids (pUC18-+ and pUC18+-) were also tested. A 1:1mixture of the two 2.9 kb plasmids containing the +- and -+ versions ofthe LacZ alpha gene (FIG. 3) was digested with DNAseI and 100-200 bpfragments were purified as described in Example 1. The shuffling programwas conducted under conditions similar to those described for reassemblyin step (1) above except the program was for 60 cycles 94° C. for 30seconds, 55° C. for 30 seconds, 72° C. for 30 seconds!. Gel analysisshowed that after the shuffling program most of the product was greaterthan 20 kb. Thus, whole 2.7 kb plasmids (pUC18 -+ and PUC18 +-) wereefficiently reassembled from random 100-200 bp fragments without addedprimers.

After digestion with a restriction enzyme having a unique site on theplasmid (EcoO109), most of the product consisted of a single band of theexpected size. This band was gel purified, religated and the DNA used totransform E. coli. The transformants were plated on 0.004% X-gal platesas described in Example 1. 11% (N=328) of the resulting plasmids wereblue and thus ++ recombinants.

3) Spiked DNA Shuffling

Oligonucleotides that are mixed into the shuffling mixture can beincorporated into the final product based on the homology of theflanking sequences of the oligonucleotide to the template DNA (FIG. 4).The LacZ⁻ stop codon mutant (pUC18 -+) described above was used as theDNAseI digested template. A 66 mer oligonucleotide, including 18 basesof homology to the wild-type LacZ gene at both ends was added into thereaction at a 4-fold molar excess to correct stop codon mutationspresent in the original gene. The shuffling reaction was conducted underconditions similar to those in step 2 above. The resulting product wasdigested, ligated and inserted into E. coli as described above.

                  TABLE 2                                                         ______________________________________                                                        % blue colonies                                               ______________________________________                                        Control            0.0 (N > 1000)                                             Top strand spike  8.0 (N = 855)                                               Bottom strand spike                                                                             9.3 (N = 620)                                               Top and bottom strand spike                                                                     2.1 (N = 537)                                               ______________________________________                                    

ssDNA appeared to be more efficient than dsDNA, presumably due tocompetitive hybridization. The degree of incorporation can be variedover a wide range by adjusting the molar excess, annealing temperature,or the length of homology.

Example 3 DNA Reassembly in the Complete Absence of Primers

Plasmid pUC18 was digested with restriction enzymes EcoRI, EcoO109, XmnIand AlwNI, yielding fragments of approximately 370, 460, 770 and 1080bp. These fragments were electrophoresed and separately purified from a2% low melting point agarose gel (the 370 and 460 basepair bands couldnot be separated), yielding a large fragment, a medium fragment and amixture of two small fragments in 3 separate tubes.

Each fragment was digested with DNAseI as described in Example 1, andfragments of 50-130 bp were purified from a 2% low melting point agarosegel for each of the original fragments.

PCR mix (as described in Example 1 above) was added to the purifieddigested fragments to a final concentration of 10 ng/μl of fragments. Noprimers were added. A reassembly reaction was performed for 75 cycles94° C. for 30 seconds, 60° C. for 30 seconds! separately on each of thethree digested DNA fragment mixtures, and the products were analyzed byagarose gel electrophoresis.

The results clearly showed that the 1080, 770 and the 370 and 460 bpbands reformed efficiently from the purified fragments, demonstratingthat shuffling does not require the use of any primers at all.

Example 4 IL-1β Gene Shuffling

This example illustrates that crossovers based on homologies of lessthan 15 bases may be obtained. As an example, a human and a murine IL-1βgene were shuffled.

A murine IL1-β gene (BBG49) and a human IL1-β gene with E. coli codonusage (BBG2; R&D Systems, Inc., Minneapolis, Minn.) were used astemplates in the shuffling reaction. The areas of complete homologybetween the human and the murine IL-1β sequences are on average only 4.1bases long (FIG. 5, regions of heterology are boxed).

Preparation of dsDNA PCR products for each of the genes, removal ofprimers, DNAseI digestion and purification of 10-50 bp fragments wassimilar to that described above in Example 1. The sequences of theprimers used in the PCR reaction were 5'TTAGGCACCCCAGGCTTT3' (SEQ IDNO:3) and 5'ATGTGCTGCAAGGCGATT3' (SEQ ID NO:4).

The first 15 cycles of the shuffling reaction were performed with theKlenow fragment of DNA polymerase I, adding 1 unit of fresh enzyme ateach cycle. The DNA was added to the PCR mix of Example 1 which mixlacked the polymerase. The manual program was 94° C. for 1 minute, andthen 15 cycles of: 95° C. for 1 minute, 10 seconds on dry ice/ethanol(until frozen), incubate about 20 seconds at 25° C. , add 1U of Klenowfragment and incubate at 25° C. for 2 minutes!. In each cycle after thedenaturation step, the tube was rapidly cooled in dry ice/ethanol andreheated to the annealing temperature. Then the heat-labile polymerasewas added. The enzyme needs to be added at every cycle. Using thisapproach, a high level of crossovers was obtained, based on only a fewbases of uninterrupted homology (FIG. 5, positions of cross-oversindicated by "₋₋ |⁻⁻ ").

After these 15 manual cycles, Taq polymerase was added and an additional22 cycles of the shuffling reaction 94° C. for 30 seconds, 35° C. for 30seconds! without primers were performed.

The reaction was then diluted 20-fold. The following primers were addedto a final concentration of 0.8 μM: 5'AACGCCGCATGCAAGCTTGGATCCTTATT3'(SEQ ID NO:5) and 5'AAAGCCCTCTAGATGATTACGAATTCATAT3' (SEQ ID NO:6) and aPCR reaction was performed as described above in Example 1. The secondprimer pair differed from the first pair only because a change inrestriction sites was deemed necessary.

After digestion of the PCR product with XbaI and SphI, the fragmentswere ligated into XbaI-SphI-digested pUC18. The sequences of the insertsfrom several colonies were determined by a dideoxy DNA sequencing kit(United States Biochemical Co., Cleveland, Ohio) according to themanufacturer's instructions.

A total of 17 crossovers were found by DNA sequencing of nine colonies.Some of the crossovers were based on only 1-2 bases of uninterruptedhomology.

It was found that to force efficient crossovers based on shorthomologies, a very low effective annealing temperature is required. Withany heat-stable polymerase, the cooling time of the PCR machine (94° C.to 25° C. at 1-2 degrees/second) causes the effective annealingtemperature to be higher than the set annealing temperature. Thus, noneof the protocols based on Taq polymerase yielded crossovers, even when aten-fold excess of one of the IL1-β genes was used. In contrast, aheat-labile polymerase, such as the Klenow fragment of DNA polymerase I,can be used to accurately obtain a low annealing temperature.

Example 5 DNA Shuffling of the TEM-1 betalactamase gene

The utility of mutagenic DNA shuffling for directed molecular evolutionwas tested in a betalactamase model system. TEM-1 betalactamase is avery efficient enzyme, limited in its reaction rate primarily bydiffusion. This example determines whether it is possible to change itsreaction specificity and obtain resistance to the drug cefotaxime thatit normally does not hydrolyze.

The minimum inhibitory concentration (MIC) of cefotaxime on bacterialcells lacking a plasmid was determined by plating 10 μl of a 10⁻²dilution of an overnight bacterial culture (about 1000 cfu) of E. coliXL1-blue cells (Stratagene, San Diego, Calif.) on plates with varyinglevels of cefotaxime (Sigma, St. Louis, Mo.), followed by incubation for24 hours at 37° C.

Growth on cefotaxime is sensitive to the density of cells, and thereforesimilar numbers of cells needed to be plated on each plate (obtained byplating on plain LB plates). Platings of 1000 cells were consistentlyperformed.

1) Initial Plasmid Construction

A pUC18 derivative carrying the bacterial TEM-1 betalactamase gene wasused (28). The TEM-1 betalactamase gene confers resistance to bacteriaagainst approximately 0.02 μg/ml of cefotaxime. Sfi1 restriction siteswere added 5' of the promoter and 3' of the end of the gene by PCR ofthe vector sequence with two primers:

Primer A (SEQ ID NO:7):5'TTCTATTGACGGCCTGTCAGGCCTCATATATACTTTAGATTGATTT3' and Primer B (SEQ IDNO:8): 5'TTGACGCACTGGCCATGGTGGCCAAAAATAAACAAATAGGGGTTCCGCGCACATTT3' andby PCR of the betalactamase gene sequence with two other primers: PrimerC (SEQ ID NO:9): 5'AACTGACCACGGCCTGACAGGCCGGTCTGACAGTTACCAATGCTT, andPrimer D (SEQ ID NO:10): 5'AACCTGTCCTGGCCACCATGGCCTAAATACATTCAAATATGTAT.

The two reaction products were digested with SfiI, mixed, ligated andused to transform bacteria.

The resulting plasmid was pUC182Sfi. This plasmid contains an Sfi1fragment carrying the TEM-1 gene and the P-3 promoter.

The minimum inhibitory concentration of cefotaxime for E. coli XL1-blue(Stratagene, San Diego, Calif.) carrying this plasmid was 0.02 μg/mlafter 24 hours at 37° C.

The ability to improve the resistance of the betalactamase gene tocefotaxime without shuffling was determined by stepwise replating of adiluted pool of cells (approximately 10⁷ cfu) on 2-fold increasing druglevels. Resistance up to 1.28 μg/ml could be obtained without shuffling.This represented a 64 fold increase in resistance.

2) DNAseI digestion

The substrate for the first shuffling reaction was dsDNA of 0.9 kbobtained by PCR of pUC182Sfi with primers C and D, both of which containa SfiI site.

The free primers from the PCR product were removed by Wizard PCR prep(Promega, Madison, Wis.) at every cycle.

About 5 μg of the DNA substrate(s) was digested with 0.15 units ofDNAseI (Sigma, St. Louis, Mo.) in 100 μl of 50 mM Tris-HCl pH 7.4, 1 mMMgCl₂, for 10 min at room temperature. Fragments of 100-300 bp werepurified from 2% low melting point agarose gels by electrophoresis ontoDE81 ion exchange paper (Whatman, Hillsborough, Oreg.), elution with 1MNaCl and ethanol precipitation by the method described in Example 1.

3) Gene shuffling

The purified fragments were resuspended in PCR mix (0.2 mM each dNTP,2.2 mM MgCl₂, 50 mM KCl, 10 mM Tris-HCl pH 9.0, 0.1% Triton X-100), at aconcentration of 10-30 ng/μl. No primers were added at this point. Areassembly program of 94° C. for 60 seconds, then 40 cycles of 94° C.for 30 seconds, 50°-55° C. for 30 seconds, 72° C. for 30 seconds! andthen 72° C. for 5 minutes was used in an MJ Research (Watertown, Mass.)PTC-150 thermocycler.

4) Amplification of Reassembly Product with primers

After dilution of the reassembly product into the PCR mix with 0.8 μM ofeach primer (C and D) and 20 PCR cycles 94° C. for 30 seconds, 50° C.for 30 seconds, 72° C. for 30 seconds! a single product 900 bp in sizewas obtained.

5) Cloning and analysis

After digestion of the 900 bp product with the terminal restrictionenzyme SfiI and agarose gel purification, the 900 bp product was ligatedinto the vector pUC182Sfi at the unique SfiI site with T4 DNA ligase(BRL, Gaithersburg, Md.). The mixture was electroporated into E. coliXL1-blue cells and plated on LB plates with 0.32-0.64 μg/ml ofcefotaxime (Sigma, St. Louis, Mo.). The cells were grown for up to 24hours at 37° C. and the resulting colonies were scraped off the plate asa pool and used as the PCR template for the next round of shuffling.

6) Subsequent Reassembly Rounds

The transformants obtained after each of three rounds of shuffling wereplated on increasing levels of cefotaxime. The colonies (>100, tomaintain diversity) from the plate with the highest level of cefotaximewere pooled and used as the template for the PCR reaction for the nextround.

A mixture of the cefotaxime^(r) colonies obtained at 0.32-0.64 μg/ml inStep (5) above were used as the template for the next round ofshuffling. 10 ul of cells in LB broth were used as the template in areassembly program of 10 minutes at 99° C., then 35 cycles of 94° C. for30 seconds, 52° C. for 30 seconds, 72° C. for 30 seconds! and then 5minutes at 72° C. as described above.

The reassembly products were digested and ligated into pUC182Sfi asdescribed in step (5) above. The mixture was electroporated into E. coliXL1-blue cells and plated on LB plates having 5-10 μg/ml of cefotaxime.

Colonies obtained at 5-10 μg/ml were used for a third round similar tothe first and second rounds except the cells were plated on LB plateshaving 80-160 μg/ml of cefotaxime. After the third round, colonies wereobtained at 80-160 μg/ml, and after replating on increasingconcentrations of cefotaxime, colonies could be obtained at up to 320μg/ml after 24 hours at 37° C. (MIC=320 μg/ml).

Growth on cefotaxime is dependent on the cell density, requiring thatall the MICs be standardized (in our case to about 1,000 cells perplate). At higher cell densities, growth at up to 1280 μg/ml wasobtained. The 5 largest colonies grown at 1,280 μg/ml were plated forsingle colonies twice, and the Sfi1 inserts were analyzed by restrictionmapping of the colony PCR products.

One mutant was obtained with a 16,000 fold increased resistance tocefotaxime (MIC=0.02 μg/ml to MIC=320 μg/ml).

After selection, the plasmid of selected clones was transferred backinto wild-type E. coli XL1-blue cells (Stratagene, San Diego, Calif.) toensure that none of the measured drug resistance was due to chromosomalmutations.

Three cycles of shuffling and selection yielded a 1.6×10⁴ -fold increasein the minimum inhibitory concentration of the extended broad spectrumantibiotic cefotaxime for the TEM-1 betalactamase. In contrast, repeatedplating without shuffling resulted in only a 16-fold increase inresistance (error-prone PCR or cassette mutagenesis).

7) Sequence analysis

All 5 of the largest colonies grown at 1,280 μg/ml had a restriction mapidentical to the wild-type TEM-1 enzyme. The SfiI insert of the plasmidobtained from one of these colonies was sequenced by dideoxy DNAsequencing (United States Biochemical Co., Cleveland, Ohio) according tothe manufacturer's instructions. All the base numbers correspond to therevised pBR322 sequence (29), and the amino acid numbers correspond tothe ABL standard numbering scheme (30). The amino acids are designatedby their three letter codes and the nucleotides by their one lettercodes. The term G4205A means that nucleotide 4205 was changed fromguanidine to adenine.

Nine single base substitutions were found. G4205A is located between the-35 and -10 sites of the betalactamase P3 promoter (31). The promoterup-mutant observed by Chen and Clowes (31) is located outside of theSfi1 fragment used here, and thus could not have been detected. Fourmutations were silent (A3689G, G3713A, G3934A and T3959A), and fourresulted in an amino acid change (C3448T resulting in Gly238Ser, A3615Gresulting in Met182Thr, C3850T resulting in Glu104Lys, and G4107Aresulting in Ala18Val).

8) Molecular Backcross

Molecular backcrossing with an excess of the wild-type DNA was then usedin order to eliminate non-essential mutations.

Molecular backcrossing was conducted on a selected plasmid from thethird round of DNA shuffling by the method identical to normal shufflingas described above, except that the DNAseI digestion and shufflingreaction were performed in the presence of a 40-fold excess of wild-typeTEM-1 gene fragment. To make the backcross more efficient, very smallDNA fragments (30 to 100 bp) were used in the shuffling reaction. Thebackcrossed mutants were again selected on LB plates with 80-160 μg/mlof cefotaxime (Sigma, St. Louis, Mo.).

This backcross shuffling was repeated with DNA from colonies from thefirst backcross round in the presence of a 40-fold excess of wild-typeTEM-1 DNA. Small DNA fragments (30-100 bp) were used to increase theefficiency of the backcross. The second round of backcrossed mutantswere again selected on LB plates with 80-160 μg/ml of cefotaxime.

The resulting transformants were plated on 160 μg/ml of cefotaxime, anda pool of colonies was replated on increasing levels of cefotaxime up to1,280 μg/ml. The largest colony obtained at 1,280 μg/ml was replated forsingle colonies.

This backcrossed mutant was 32,000 fold more resistant than wild-type.(MIC=640 μg/ml) The mutant strain is 64-fold more resistant tocefotaxime than previously reported clinical or engineered TEM-1-derivedstrains. Thus, it appears that DNA shuffling is a fast and powerful toolfor at least several cycles of directed molecular evolution.

The DNA sequence of the SfiI insert of the backcrossed mutant wasdetermined using a dideoxy DNA sequencing kit (United States BiochemicalCo., Cleveland, Ohio) according to the manufacturer's instructions(Table 3). The mutant had 9 single base pair mutations. As expected, allfour of the previously identified silent mutations were lost, revertingto the sequence of the wild-type gene. The promoter mutation (G4205A) aswell as three of the four amino acid mutations (Glu104Lys, Met182Thr,and Gly238Ser) remained in the backcrossed clone, suggesting that theyare essential for high level cefotaxime resistance. However, two newsilent mutations (T3842C and A3767G), as well as three new mutationsresulting in amino acid changes were found (C3441T resulting inArg241His, C3886T resulting in Gly92Ser, and G4035C resulting inAla42Gly). While these two silent mutations do not affect the proteinprimary sequence, they may influence protein expression level (forexample by mRNA structure) and possibly even protein folding (bychanging the codon usage and therefore the pause site, which has beenimplicated in protein folding).

                  TABLE 3                                                         ______________________________________                                        Mutations in Betalactamase                                                    Mutation Type Non-Backcrossed                                                                             Backcrossed                                       ______________________________________                                        amino acid    Ala18Lys      --                                                change        Glu104Lys     Glu104Lys                                                       Met182Thr     Met182Thr                                                       Gly238Ser     Gly238Ser                                                       --            Ala42Gly                                                        --            Gly92Ser                                          silent        T3959A        --                                                              G3934A        --                                                              G3713A        --                                                              A3689G        --                                                              --            T3842C                                                          --            A3767G                                            promoter      G4205A        G4205A                                            ______________________________________                                    

Both the backcrossed and the non-backcrossed mutants have a promotermutation (which by itself or in combination results in a 2-3 foldincrease in expression level) as well as three common amino acid changes(Glu104Lys, Met182Thr and Gly238Ser). Glu104Lys and Gly238Ser aremutations that are present in several cefotaxime resistant or otherTEM-1 derivatives (Table 4).

9) Expression Level Comparison

The expression level of the betalactamase gene in the wild-type plasmid,the non-backcrossed mutant and in the backcrossed mutant was compared bySDS-polyacrylamide gel electrophoresis (4-20%; Novex, San Diego, Calif.)of periplasmic extracts prepared by osmotic shock according to themethod of Witholt, B. (32).

Purified TEM-1 betalactamase (Sigma, St. Louis, Mo.) was used as amolecular weight standard, and E. coli XL1-blue cells lacking a plasmidwere used as a negative control.

The mutant and the backcrossed mutant appeared to produce a 2-3 foldhigher level of the betalactamase protein compared to the wild-typegene. The promoter mutation appeared to result in a 2-3 times increasein betalactamase.

Example 6 Construction of Mutant Combinations of the TEM-1 BetalactamaseGene

To determine the resistance of different combinations of mutations andto compare the new mutants to published mutants, several mutants wereconstructed into an identical plasmid background. Two of the mutations,Glu104Lys and Gly238Ser, are known as cefotaxime mutants. All mutantcombinations constructed had the promoter mutation, to allow comparisonto selected mutants. The results are shown in Table 4.

Specific combinations of mutations were introduced into the wild-typepUC182Sfi by PCR, using two oligonucleotides per mutation.

The oligonucleotides to obtain the following mutations were: ##STR1##

These separate PCR fragments were gel purified away from the syntheticoligonucleotides. 10 ng of each fragment were combined and a reassemblyreaction was performed at 94° C. for 1 minute and then 25 cycles; 94° C.for 30 sec, 50° C. for 30 seconds and 72° C. for 45 seconds). PCR wasperformed on the reassembly product for 25 cycles in the presence of theSfiI-containing outside primers (primers C and D from Example 5). TheDNA was digested with Sfi1 and inserted into the wild-type pUC182Sfivector. The following mutant combinations were obtained (Table 4).

                  TABLE 4                                                         ______________________________________                                                                           Source                                     Name   Genotype            MIC     of MIC                                     ______________________________________                                        TEM-1  Wild-type           0.02                                                      Glu104Lys           0.08    10                                                Gly238Ser           016     10                                         TEM-15 Glu104Lys/Gly238Ser*                                                                              10                                                 TEM-3  Glu104Lys/Gly238Ser/Gln39Lys                                                                      102-32  37, 15                                     ST-4   Glu104Lys/Gly238Ser/Met182Thr*                                                                    10                                                 ST-1   Glu104Lys/Gly238Ser/Met182Thr/                                                                    320                                                       Ala18Val/T3959A/G3713A/G3934A/                                                A3689G*                                                                ST-2   Glu104Lys/Gly238Ser/Met182Thr/                                                                    640                                                       Ala42Gly/Gly92Ser/Arg241His/                                                  T3842C/A3767G*                                                         ST-3   Glu104Lys/Gly238Ser/Met182Thr/                                                                    640                                                       Ala42Gly/Gly92Ser/Arg241His*                                           ______________________________________                                         *All of these mutants additionally contain the G4205A promoter mutation. 

It was concluded that conserved mutations account for 9 of 15 doublingsin the MIC.

Glu104Lys alone was shown to result only in a doubling of the MIC to0.08 μg/ml, and Gly238Ser (in several contexts with one additional aminoacid change) resulted only in a MIC of 0.16 μg/ml (26). The doublemutant Glu104Lys/Gly238Ser has a MIC of 10 μg/ml. This mutantcorresponds to TEM-15.

These same Glu104Lys and Gly238Ser mutations, in combination withGln39Lys (TEM-3) or Thr263Met (TEM-4) result in a high level ofresistance (2-32 μg/ml for TEM-3 and 8-32 μg/ml for TEM-4 (34, 35).

A mutant containing the three amino acid changes that were conservedafter the backcross (Glu104Lys/Met182Thr/Gly238Ser) also had a MIC of 10μg/ml. This meant that the mutations that each of the new selectedmutants had in addition to the three known mutations were responsiblefor a further 32 to 64-fold increase in the resistance of the gene tocefotaxime.

The naturally occurring, clinical TEM-l-derived enzymes (TEM-1-19) eachcontain a different combination of only 5-7 identical mutations(reviews). Since these mutations are in well separated locations in thegene, a mutant with high cefotaxime resistance cannot be obtained bycassette mutagenesis of a single area. This may explain why the maximumMIC that was obtained by the standard cassette mutagenesis approach isonly 0.64 μg/ml (26). For example, both the Glu104Lys as well as theGly238Ser mutations were found separately in this study to have MICsbelow 0.16 μg/ml. Use of DNA shuffling allowed combinatoriality and thusthe Glu104Lys/Gly238Ser combination was found, with a MIC of 10 μg/ml.

An important limitation of this example is the use of a single gene as astarting point. It is contemplated that better combinations can be foundif a large number of related, naturally occurring genes are shuffled.The diversity that is present in such a mixture is more meaningful thanthe random mutations that are generated by mutagenic shuffling. Forexample, it is contemplated that one could use a repertoire of relatedgenes from a single species, such as the pre-existing diversity of theimmune system, or related genes obtained from many different species.

Example 7 Improvement of Antibody A10B by DNA Shuffling of a Library ofAll Six Mutant CDRs

The A10B scFv antibody, a mouse anti-rabbit IgG, was a gift fromPharmacia (Milwaukee, Wis.). The commercially available Pharmacia phagedisplay system was used, which uses the pCANTAB5 phage display vector.

The original A10B antibody reproducibly had only a low avidity, sinceclones that only bound weakly to immobilized antigen (rabbit IgG), (asmeasured by phage ELISA (Pharmacia assay kit) or by phage titer) wereobtained. The concentration of rabbit IgG which yielded 50% inhibitionof the A10B antibody binding in a competition assay was 13 picomolar.The observed low avidity may also be due to instability of the A10Bclone.

The A10B scFv DNA was sequenced (United States Biochemical Co.,Cleveland, Ohio) according to the manufacturer's instructions. Thesequence was similar to existing antibodies, based on comparison toKabat (33).

1) Preparation of phage DNA

Phage DNA having the A10B wild-type antibody gene (10 ul) was incubatedat 99° C. for 10 min, then at 72° C. for 2 min. PCR mix (50 mM KCl, 10mM Tris-HCl pH 9.0, 0.1% Triton X-100, 200 μM each dNTP, 1.9 mM MgCl),0.6 μm of each primer and 0.5 μl Taq DNA Polymerase (Promega, Madison,Wis.) was added to the phage DNA. A PCR program was run for 35 cycles of30 seconds at 94° C., 30 seconds at 45° C., 45 seconds at 72° C.!. Theprimers used were: 5' ATGATTACGCCAAGCTTT 3' (SEQ ID NO:26) and 5'TTGTCGTCTTTCCAGACGTT 3' (SEQ ID NO:27).

The 850 bp PCR product was then electrophoresed and purified from a 2%low melting point agarose gel.

2) Fragmentation

300 ng of the gel purified 850 bp band was digested with 0.18 units ofDNAse I (Sigma, St. Louis, Mo.) in 50 mM Tris-HCl pH 7.5, 10 mM MgCl for20 minutes at room temperature. The digested DNA was separated on a 2%low melting point agarose gel and bands between 50 and 200 bp werepurified from the gel.

3) Construction of Test Library

The purpose of this experiment was to test whether the insertion of theCDRs would be efficient.

The following CDR sequences having internal restriction enzyme siteswere synthesized. "CDR H" means a CDR in the heavy chain and "CDR L"means a CDR in the light chain of the antibody. ##STR2##

The CDR oligos were added to the purified A10B antibody DNA fragments ofbetween 50 to 200 bp from step (2) above at a 10 fold molar excess. ThePCR mix (50 mM KCl, 10 mM Tris-HCl pH 9.0, 0.1% Triton x-100, 1.9 mMMgCl, 200 μm each dNTP, 0.3 μl Taq DNA polymerase (Promega, Madison,Wis.), 50 μl total volume) was added and the shuffling program run for 1min at 94° C., 1 min at 72° C., and then 35 cycles: 30 seconds at 94°C., 30 seconds at 55° C., 30 seconds at 72° C.

1 μl of the shuffled mixture was added to 100 μl of a PCR mix (50 mMKCl, 10 mM Tris-HCl pH 9.0, 0.1% Triton X-100, 200 μm each dNTP, 1.9 mMMgCl, 0.6 μM each of the two outside primers (SEQ ID NO;26 and 27, seebelow), 0.5 μl Taq DNA polymerase) and the PCR program was run for 30cycles of 30 seconds at 94° C., 30 seconds at 45° C., 45 seconds at 72°C.!. The resulting mixture of DNA fragments of 850 basepair size wasphenol/chloroform extracted and ethanol precipitated.

The outside primers were:

Outside Primer 1: SEQ ID NO:27 5' TTGTCGTCTTTCCAGACGTT 3'

Outside Primer 2: SEQ ID NO:26 5' ATGATTACGCCAAGCTTT 3'

The 850 bp PCR product was digested with the restriction enzymes SfiIand NotI, purified from a low melting point agarose gel, and ligatedinto the pCANTAB5 expression vector obtained from Pharmacia, Milwaukee,Wis. The ligated vector was electroporated according to the method setforth by Invitrogen (San Diego, Calif.) into TG1 cells (Pharmacia,Milwaukee, Wis.) and plated for single colonies.

The DNA from the resulting colonies was added to 100 μl of a PCR mix (50mM KCl, 10 mM Tris-HCl pH 9.0, 0.1% Triton X-100, 200 μm each dNTP, 1.9mM MgCl, 0.6 μM of Outside primer 1 (SEQ ID No. 27; see below) sixinside primers (SEQ ID NOS:40-45; see below), and 0.5 μl Taq DNApolymerase) and a PCR program was run for 35 cycles of 30 seconds at 94°C., 30 seconds at 45° C., 45 seconds at 72° C.!. The sizes of the PCRproducts were determined by agarose gel electrophoresis, and were usedto determine which CDRs with restriction sites were inserted.

CDR Inside Primers: ##STR3##

The six synthetic CDRs were inserted at the expected locations in thewild-type A10B antibody DNA (FIG. 7). These studies showed that, whileeach of the six CDRs in a specific clone has a small chance of being aCDR with a restriction site, most of the clones carried at least one CDRwith a restriction site, and that any possible combination of CDRs withrestriction sites was generated.

4) Construction of Mutant Complementarity Determining Regions ("CDRs")

Based on our sequence data six oligonucleotides corresponding to the sixCDRs were made. The CDRs (Kabat definition) were syntheticallymutagenized at a ratio of 70 (existing base):10:10:10, and were flankedon the 5' and 3' sides by about 20 bases of flanking sequence, whichprovide the homology for the incorporation of the CDRs when mixed into amixture of unmutagenized antibody gene fragments in a molar excess. Theresulting mutant sequences are given below. ##STR4## Bold and underlinedsequences were the mutant sequences synthesized using a mixture ofnucleosides of 70:10:10:10 where 70% was the wild-type nucleoside.

A 10 fold molar excess of the CDR mutant oligos were added to thepurified A10B antibody DNA fragments between 50 to 200 bp in length fromstep (2) above. The PCR mix (50 mM KCl, 10 mM Tris-HCl pH 9.0, 0.1%Triton x-100, 1.9 mM MgCl, 200 μm each dNTP, 0.3 μl Taq DNA polymerase(Promega, Madison, Wis.), 50 μl total volume) was added and theshuffling program run for 1 min at 94° C., 1 min at 72° C., and then 35cycles: 30 seconds at 94° C., 30 seconds at 55° C., 30 seconds at 72°C.!.

1 μl of the shuffled mixture was added to 100 μl of a PCR mix (50 mMKCl, 10 mM Tris-HCl pH 9.0, 0.1% Triton X-100, 200 μm each dNTP, 1.9 mMMgCl, 0.6 μM each of the two outside primers (SEQ ID NO:26 and 27, seebelow), 0.5 μl Taq DNA polymerase) and the PCR program was run for 30cycles of (30 seconds at 94° C., 30 seconds at 45° C., 45 seconds at 72°C.!. The resulting mixture of DNA fragments of 850 basepair size wasphenol/chloroform extracted and ethanol precipitated.

The outside primers were:

Outside Primer 1: SEQ ID NO:27 5' TTGTCGTCTTTCCAGACGTT 3'

Outside Primer 2: SEQ ID NO:26 5' ATGATTACGCCAAGCTTT 3'

5) Cloning of the scFv antibody DNA into pCANTAB5

The 850 bp PCR product was digested with the restriction enzymes SfiIand NotI, purified from a low melting point agarose gel, and ligatedinto the pCANTAB5 expression vector obtained from Pharmacia, Milwaukee,Wis. The ligated vector was electroporated according to the method setforth by Invitrogen (San Diego, Calif.) into TG1 cells (Pharmacia,Milwaukee, Wis.) and the phage library was grown up using helper phagefollowing the guidelines recommended by the manufacturer.

The library that was generated in this fashion was screened for thepresence of improved antibodies, using six cycles of selection.

6) Selection of high affinity clones

15 wells of a 96 well microtiter plate were coated with Rabbit IgG(Jackson Immunoresearch, Bar Harbor, Me.) at 10 μg/well for 1 hour at37° C., and then blocked with 2% non-fat dry milk in PBS for 1 hour at37° C.

100 μl of the phage library (1×10¹⁰ cfu) was blocked with 100 μl of 2%milk for 30 minutes at room temperature, and then added to each of the15 wells and incubated for 1 hour at 37° C.

Then the wells were washed three times with PBS containing 0.5% Tween-20at 37° C. for 10 minutes per wash. Bound phage was eluted with 100 μlelution buffer (Glycine-HCl, pH 2.2), followed by immediateneutralization with 2M Tris pH 7.4 and transfection for phageproduction. This selection cycle was repeated six times.

After the sixth cycle, individual phage clones were picked and therelative affinities were compared by phage ELISA, and the specificityfor the rabbit IgG was assayed with a kit from Pharmacia (Milwaukee,Wis.) according to the methods recommended by the manufacturer.

The best clone has an approximately 100-fold improved expression levelcompared with the wild-type A10B when tested by the Western assay. Theconcentration of the rabbit IgG which yielded 50% inhibition in acompetition assay with the best clone was 1 picomolar. The best clonewas reproducibly specific for rabbit antigen. The number of copies ofthe antibody displayed by the phage appears to be increased.

Example 8 In vivo Recombination Via Direct Repeats of Partial Genes

A plasmid was constructed with two partial, inactive copies of the samegene (beta-lactamase) to demonstrate that recombination between thecommon areas of these two direct repeats leads to full-length, activerecombinant genes.

A pUC18 derivative carrying the bacterial TEM-1 betalactamase gene wasused (Yanish-Perron et al., 1985, Gene 33:103-119). The TEM-1betalactamase gene ("Bla") confers resistance to bacteria againstapproximately 0.02 μg/ml of cefotaxime. Sfi1 restriction sites wereadded 51 of the promoter and 3' of the end of the betalactamase gene byPCR of the vector sequence with two primers:

Primer A (SEQ ID NO: 46) 5'TTCTATTGACGGCCTGTCAGGCCTCATATATACTTTAGATTGATTT 3'

PRIMER B (SEQ ID NO: 47) 5'TTGACGCACTGGCCATGGTGGCCAAAAATAAACAAATAGGGGTTCCGCGCAC ATTT 3'

and by PCR of the beta-lactamase gene sequence with two other primers:

Primer C (SEQ ID NO: 48) 5'AACTGACCACGGCCTGACAGGCCGGTCTGACAGTTACCAATGCTT 3'

Primer D (SEQ ID NO: 49) 5' AACCTGTCCTGGCCACCATGGCCTAAATACATTCAAATATGTAT3'

The two reaction products were digested with Sfi1, mixed, ligated andused to transform competent E. coli bacteria by the procedure describedbelow. The resulting plasmid was pUC182Sfi-Bla-Sfi. This plasmidcontains an Sfi1 fragment carrying the Bla gene and the P-3 promoter.

The minimum inhibitory concentration of cefotaxime for E. coli XL1-blue(Stratagene, San Diego, Calif.) carrying pUC182Sfi-Bla-Sfi was 0.02μg/ml after 24 hours at 37° C.

The tetracycline gene of pBR322 was cloned into pUC18Sfi-Bla-Sfi usingthe homologous areas, resulting in pBR322TetSfi-Bla-Sfi. The TEM-1 genewas then deleted by restriction digestion of the pBR322TetSfi-Bla-Sfiwith SspI and FspI and blunt-end ligation, resulting inpUC322TetSfi-Sfi.

Overlapping regions of the TEM-1 gene were amplified using standard PCRtechniques and the following primers:

Primer 2650 (SEQ ID NO: 50) 5' TTCTTAGACGTCAGGTGGCACTT 3'

Primer 2493 (SEQ ID NO: 51) 5' TTT TAA ATC AAT CTA AAG TAT 3'

Primer 2651 (SEQ ID NO: 52) 5' TGCTCATCCACGAGTGTGGAGAAGTGGTCCTGCAACTTTAT3,' and

Primer 2652 (SEQ ID NO: 53) ACCACTTCTCCACACTCGTGGATGAGCACTTTTAAAGTT

The two resulting DNA fragments were digested with Sfi1 and BstX1 andligated into the Sfi site of pBR322TetSfi-Sfi. The resulting plasmid wascalled pBR322Sfi-BL-LA-Sfi. A map of the plasmid as well as a schematicof intraplasmidic recombination and reconstitution of functionalbeta-lactamase is shown in FIG. 9.

The plasmid was electroporated into either TG-1 or JC8679 E. coli cells.E. coli JC8679 is RecBC sbcA (Oliner et al., 1993, NAR 21:5192). Thecells were plated on solid agar plates containing tetracycline. Thosecolonies which grew, were then plated on solid agar plates containing100 μg/ml ampicillin and the number of viable colonies counted. Thebeta-lactamase gene inserts in those transformants which exhibitedampicillin resistance were amplified by standard PCR techniques usingPrimer 2650 (SEQ ID NO: 50) 5' TTCTTAGACGTCAGGTGGCACTT 3' and Primer2493 (SEQ ID NO: 51) 5' TTTTAAATCAATCTAAAGTAT 3' and the length of theinsert measured. The presence of a 1 kb insert indicates that the genewas successfully recombined, as shown in FIG. 9 and Table 5.

                  TABLE 5                                                         ______________________________________                                        Cell        Tet Colonies                                                                            Amp colonies Colony PCR                                 ______________________________________                                        TG-1        131       21           3/3 at 1 kb                                JC8679      123       31           4/4 at 1 kb                                vector control                                                                             .sup. 5I  0                                                      ______________________________________                                    

About 17-25% of the tetracycline-resistant colonies were alsoampicillin-resistant and all of the Ampicillin resistant colonies hadcorrectly recombined, as determined by colony PCR. Therefore, partialgenes located on the same plasmid will successfully recombine to createa functional gene.

Example 9 In vivo Recombination Via Direct Repeats of Full-Length Genes

A plasmid with two full-length copies of different alleles of thebeta-lactamase gene was constructed. Homologous recombination of the twogenes resulted in a single recombinant full-length copy of that gene.

The construction of pBR322TetSfi-Sfi and pBR322TetSfi-Bla-Sfi wasdescribed above.

The two alleles of the beta-lactamase gene were constructed as follows.Two PCR reactions were conducted with pUC18Sfi-Bla-Sfi as the template.One reaction was conducted with the following primers.

Primer 2650 (SEQ ID NO: 50) 5' TTCTTAGACGTCAGGTGGCACTT 3'

Primer 2649 (SEQ ID NO: 51) 5'ATGGTAGTCCACGAGTGTGGTAGTGACAGGCCGGTCTGACAGTTA CCAATGCTT 3'

The second PCR reaction was conducted with the following primers:

Primer 2648 (SEQ ID NO: 54) 5'TGTCACTACCACACTCGTGGACTACCATGGCCTAAATACATTCAAA TATGTAT 3'

Primer 2493 (SEQ ID NO: 51) 5' TTT TAA ATC AAT CTA AAG TAT 3'

This yielded two Bla genes, one with a 5' Sfi1 site and a 3' BstX1 site,the other with a 5' BstX1 site and a 3' Sfi1 site.

After digestion of these two genes with BstX1 and Sfi1, and ligationinto the Sfi1-digested plasmid pBR322TetSfi-Sfi, a plasmid(pBR322-Sfi-2BLA-Sfi) with a tandem repeat of the Bla gene was obtained.(See FIG. 10).

The plasmid was electroporated into E. coli cells. The cells were platedon solid agar plates containing 15 μg/ml tetracycline. Those colonieswhich grew, were then plated on solid agar plates containing 100 μg/mlampicillin and the number of viable colonies counted. The Bla inserts inthose transformants which exhibited ampicillin resistance were amplifiedby standard PCR techniques using the method and primers described inExample 8. The presence of a 1 kb insert indicated that the duplicategenes had recombined, as indicated in Table 6.

                  TABLE 6                                                         ______________________________________                                        Cell        Tet Colonies                                                                            Amp Colonies Colony PCR                                 ______________________________________                                        TG-1         28        54          7/7 at 1 kb                                JC8679      149       117          3/3 at 1 kb                                vector control                                                                             51        0                                                      ______________________________________                                    

Colony PCR confirmed that the tandem repeat was efficiently recombinedto form a single recombinant gene.

Example 10 Multiple Cycles of Direct RepeatRecombination--Interplasmidic

In order to determine whether multiple cycles of recombination could beused to produce resistant cells more quickly, multiple cycles of themethod described in Example 9 were performed.

The minus recombination control consisted of a single copy of thebetalactamase gene, whereas the plus recombination experiment consistedof inserting two copies of betalactamase as a direct repeat. Thetetracycline marker was used to equalize the number of colonies thatwere selected for cefotaxime resistance in each round, to compensate forligation efficiencies.

In the first round, pBR322TetSfi-Bla-Sfi was digested with EcrI andsubject to PCR with a 1:1 mix (1 ml) of normal and Cadwell PCR mix(Cadwell and Joyce (1992) PCR Methods and Applications 2: 28-33) forerror prone PCR. The PCR program was 70° C. for 2 minutes initially andthen 30 cycles of 94° C. for 30 seconds, 52° C. for 30 second and 72° C.for 3 minutes and 6 seconds per cycle, followed by 72° C. for 10minutes.

The primers used in the PCR reaction to create the one Bla gene controlplasmid were Primer 2650 (SEQ ID NO: 50) and Primer 2719 (SEQ ID NO: 55)5' TTAAGGGATTTTGGTCATGAGATT 3'. This resulted in a mixed population ofamplified DNA fragments, designated collectively as Fragment #59. Thesefragments had a number of different mutations.

The primers used in two different PCR reactions to create the two Blagene plasmid were Primer 2650 (SEQ ID NO: 50) and Primer 2649 (SEQ IDNO: 51) for the first gene and Primers 2648 (SEQ ID NO: 54) and Primer2719 (SEQ ID NO: 55) for the second gene. This resulted in a mixedpopulation of each of the two amplified DNA fragments: Fragment #89(amplified with primers 2648 and 2719) and Fragment #90 (amplified withprimers 2650 and 2649). In each case a number of different mutations hadbeen introduced the mixed population of each of the fragments.

After error prone PCR, the population of amplified DNA fragment #59 wasdigested with Sfi1, and then cloned into pBR322TetSfi-Sfi to create amixed population of the plasmid pBR322Sfi-Bla-Sfi¹.

After error prone PCR, the population of amplified DNA fragments #90 and#89 was digested with SfiI and BstXI at 50° C., and ligated intopBR322TetSfi-Sfi to create a mixed population of the plasmidpBR322TetSfi-2Bla-Sfi¹ (FIG. 10).

The plasmids pBR322Sfi-Bla-Sfi¹ and pBR322Sfi-2Bla-Sfi¹ wereelectroporated into E. coli JC8679 and placed on agar plates havingdiffering concentrations of cefotaxime to select for resistant strainsand on tetracycline plates to titre.

An equal number of colonies (based on the number of colonies growing ontetracycline) were picked, grown in LB-tet and DNA extracted from thecolonies. This was one round of the recombination. This DNA was digestedwith EcrI and used for a second round of error-prone PCR as describedabove.

After five rounds the MIC (minimum inhibitory concentration) forcefotaxime for the one fragment plasmid was 0.32 whereas the MIC for thetwo fragment plasmid was 1.28. The results show that after five cyclesthe resistance obtained with recombination was four-fold higher in thepresence of in vivo recombination.

Example 11 In vivo Recombination Via Electroporation of Fragments

Competent E. coli cells containing pUC18Sfi-Bla-Sfi were prepared asdescribed. Plasmid pUC18Sfi-Bla-Sfi contains the standard TEM-1beta-lactamase gene as described, supra.

A TEM-1 derived cefotaxime resistance gene from pUC18Sfi-cef-Sfi, (cloneST2) (Stemmer, W. P. C (1994) Nature 370: 389-91, incorporated herein byreference) which confers on E. coli carrying the plasmid an MIC of 640μg/ml for cefotaxime, was obtained. In one experiment the completeplasmid pUC18Sfi-cef-Sfi DNA was electroporated into E. coli cellshaving the plasmid pUC18Sfi-Bla-Sfi.

In another experiment the DNA fragment containing the cefotaxime genefrom pUC18Sfi-cef-Sfi was amplified by PCR using the primers 2650 (SEQID NO: 50) and 2719 (SEQ ID NO: 55). The resulting 1 kb PCR product wasdigested into DNA fragments of <100 bp by DNase and these fragments wereelectroporated into the competent E. coli cells which already containedpUC18Sfi-Bla-Sfi.

The transformed cells from both experiments were then assayed for theirresistance to cefotaxime by plating the transformed cells onto agarplates having varying concentrations of cefotaxime. The results areindicated in Table 7.

                  TABLE 7                                                         ______________________________________                                                    Colonies/Cefotaxime Concentration                                             0.16 0.32     1.28   5.0    10.0                                  ______________________________________                                        no DNA control                                                                              14                                                              ST-2 mutant, whole   4000     2000 800    400                                 ST-2 mutant, fragments                                                                             1000      120  22     7                                  Wildtype, whole                                                                             27                                                              Wildtype, fragments                                                                         18                                                              ______________________________________                                    

From the results it appears that the whole ST-2 Cef gene was insertedinto either the bacterial genome or the plasmid after electroporation.Because most insertions are homologous, it is expected that the gene wasinserted into the plasmid, replacing the wildtype gene. The fragments ofthe Cef gene from St-2 also inserted efficiently into the wild-type genein the plasmid. No sharp increase in cefotaxime resistance was observedwith the introduction of the wildtype gene (whole or in fragments) andno DNA. Therefore, the ST-2 fragments were shown to yield much greatercefotaxime resistance than the wild-type fragments. It was contemplatedthat repeated insertions of fragments, prepared from increasingresistant gene pools would lead to increasing resistance.

Accordingly, those colonies that produced increased cefotaximeresistance with the St-2 gene fragments were isolated and the plasmidDNA extracted. This DNA was amplified using PCR by the method describedabove. The amplified DNA was digested with DNase into fragments (<100bp) and 2-4 μg of the fragments were electroporated into competent E.coli cells already containing pUC322Sfi-Bla-Sfi as described above. Thetransformed cells were plated on agar containing varying concentrationsof cefotaxime.

As a control, competent E. coli cells having the plasmidpUC18Sfi-Kan-Sfi were also used. DNA fragments from the digestion of thePCR product of pUC18Sfi-cef-Sfi were electroporated into these cells.There is no homology between the kanamycin gene and the beta-lactamasegene and thus recombination should not occur.

This experiment was repeated for 2 rounds and the results are shown inTable 8.

                  TABLE 8                                                         ______________________________________                                                                        Cef resistant                                 Round   Cef conc.   KAN control colonies                                      ______________________________________                                        1       0.16-0.64          lawn   lawn                                        replate 0.32        10     small  1000                                        2       10          10             400                                        Replate             100    sm @ 2.5                                                                             50 @ 10                                     3       40          100    sm                                                         1280                      100 sm                                      ______________________________________                                    

Example 12 Determination of Recombination Formats

This experiment was designed to determine which format of recombinationgenerated the most recombinants per cycle.

In the first approach, the vector pUC18Sfi-Bla-Sfi was amplified withPCR primers to generate a large and small fragment. The large fragmenthad the plasmid and ends having portions of the Bla gene, and the smallfragment coded for the middle of the Bla gene. A third fragment havingthe complete Bla gene was created using PCR by the method in Example 8.The larger plasmid fragment and the fragment containing the complete Blagene were electroporated into E. coli JC8679 cells at the same time bythe method described above and the transformants plated on differingconcentrations of cefotaxime.

In approach 2, the vector pUC18Sfi-Bla-Sfi was amplified to produce thelarge plasmid fragment isolated as in approach 1 above. The twofragments each comprising a portion of the complete Bla gene, such thatthe two fragments together spanned the complete Bla gene werealsoobtained by PCR. The large plasmid fragment and the two Bla genefragments were all electroporated into competent E. coli JC8679 cellsand the transformants plated on varying concentrations of cefotaxime.

In the third approach, both the vector and the plasmid wereelectroporated into E. coli JC8679 cells and the transformants wereplated on varying concentrations of cefotaxime.

In the fourth approach, the complete Bla gene was electroporated into E.coli JC8679 cells already containing the vector pUCSfi-Sfi and thetransformants were plated on varying concentrations of cefotaxime. Ascontrols, the E. coli JC8679 cells were electroporated with either thecomplete Bla gene or the vector alone.

The results are presented in FIG. 11. The efficiency of the insertion oftwo fragments into the vector is 100 X lower than when one fragmenthaving the complete Bla gene is used. Approach 3 indicated that theefficiency of insertion does depend on the presence of free DNA endssince no recombinants were obtained with this approach. However, theresults of approach 3 were also due to the low efficiency ofelectroporation of the vector. When the expression vector is already inthe competent cells, the efficiency of the vector electroporation is notlonger a factor and efficient homologous recombination can be achievedeven with uncut vector.

Example 12 Kit for Cassette Shuffling to Optimize Vector Performance

In order to provide a vector capable of conferring an optimizedphenotype (e.g., maximal expression of a vector-encoded sequence, suchas a cloned gene), a kit is provided comprising a variety of cassetteswhich can be shuffled, and optimized shufflants can be selected. FIG. 12shows schematically one embodiment, with each loci having a plurality ofcassettes. For example, in a bacterial expression system, FIG. 13 showsexample cassettes that are used at the respective loci. Each cassette ofa given locus (e.g., all promoters in this example) are flanked bysubstantially identical sequences capable of overlapping the flankingsequence(s) of cassettes of an adjacent locus and preferably alsocapable of participating in homologous recombination or non-homologousrecombination (e.g., lox/cre or flp/frt systems), so as to affordshuffling of cassettes within a locus but substantially not betweenloci.

Cassettes are supplied in the kit as PCR fragments, which each cassettetype or individual cassette species packaged in a separate tube. Vectorlibraries are created by combining the contents of tubes to assemblewhole plasmids or substantial portions thereof by hybridization of theoverlapping flanking sequences of cassettes at each locus with cassettesat the adjacent loci. The assembled vector is ligated to a predeterminedgene of interest to form a vector library wherein each library membercomprises the predetermined gene of interest and a combination ofcassettes determined by the association of cassettes. The vectors aretransferred into a suitable host cell and the cells are cultured underconditions suitable for expression, and the desired phenotype isselected.

While the present invention has been described with reference to whatare considered to be the preferred examples, it is to be understood thatthe invention is not limited to the disclosed examples. To the contrary,the invention is intended to cover various modifications and equivalentarrangements included within the spirit and scope of the appendedclaims.

REFERENCES

The following references are cited in this application at the relevantportion of the application.

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All publications, patents and patent applications are hereinincorporated by reference in their entirety to the same extent as ifeach individual publication, patent or patent application wasspecifically and individually indicated to be incorporated by referencein its entirety.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 62                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       AAAGCGTCGATTTTTGTGAT20                                                        (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       ATGGGGTTCCGCGCACATTT20                                                        (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       TTAGGCACCCCAGGCTTT18                                                          (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       ATGTGCTGCAAGGCGATT18                                                          (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 29 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       AACGCCGCATGCAAGCTTGGATCCTTATT29                                               (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       AAAGCCCTCTAGATGATTACGAATTCATAT30                                              (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 46 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       TTCTATTGACGGCCTGTCAGGCCTCATATATACTTTAGATTGATTT46                              (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 56 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       TTGACGCACTGGCCATGGTGGCCAAAAATAAACAAATAGGGGTTCCGCGCACATTT56                    (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 45 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       AACTGACCACGGCCTGACAGGCCGGTCTGACAGTTACCAATGCTT45                               (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 44 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      AACCTGTCCTGGCCACCATGGCCTAAATACATTCAAATATGTAT44                                (2) INFORMATION FOR SEQ ID NO:11:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 31 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                      AGTTGGGTGGACGAGTGGGTTACATCGAACT31                                             (2) INFORMATION FOR SEQ ID NO:12:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 33 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                      AACCCACTCGTCCACCCAACTGATCTTCAGCAT33                                           (2) INFORMATION FOR SEQ ID NO:13:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 41 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                      AGTAAAAGATGCTGAAGATAAGTTGGGTGCACGAGTGGGTT41                                   (2) INFORMATION FOR SEQ ID NO:14:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                      ACTTATCTTCAGCATCTTTTACTT24                                                    (2) INFORMATION FOR SEQ ID NO:15:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 32 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                      AAGAGCAACTCAGTCGCCGCATACACTATTCT32                                            (2) INFORMATION FOR SEQ ID NO:16:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 36 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                      ATGGCGGCGACTGAGTTGCTCTTGCCCGGCGTCAAT36                                        (2) INFORMATION FOR SEQ ID NO:17:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 42 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                      TATTCTCAGAATGACTTGGTTAAGTACTCACCAGTCACAGAA42                                  (2) INFORMATION FOR SEQ ID NO:18:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                      TTAACCAAGTCATTCTGAGAAT22                                                      (2) INFORMATION FOR SEQ ID NO:19:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 36 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                                      AACGACGAGCGTGACACCACGACGCCTGTAGCAATG36                                        (2) INFORMATION FOR SEQ ID NO:20:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                                      TCGTGGTGTCACGCTCGTCGTT22                                                      (2) INFORMATION FOR SEQ ID NO:21:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 42 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                                      TTGCTGATAAATCTGGAGCCAGTGAGCGTGGGTCTCGCGGTA42                                  (2) INFORMATION FOR SEQ ID NO:22:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                                      TGGCTCCAGATTTATCAGCAA21                                                       (2) INFORMATION FOR SEQ ID NO:23:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                                      ATGCTCACTGGCTCCAGATTTATCAGCAAT30                                              (2) INFORMATION FOR SEQ ID NO:24:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 36 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:                                      TCTGGAGCCAGTGAGCATGGGTCTCGCGGTATCATT36                                        (2) INFORMATION FOR SEQ ID NO:25:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 70 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:                                      AACCTGTCCTGGCCACCATGGCCTAAATACAATCAAATATGTATCCGCTTATGAGACAAT60                AACCCTGATA70                                                                  (2) INFORMATION FOR SEQ ID NO:26:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:                                      ATGATTACGCCAAGCTTT18                                                          (2) INFORMATION FOR SEQ ID NO:27:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:                                      TTGTCGTCTTTCCAGACGTT20                                                        (2) INFORMATION FOR SEQ ID NO:28:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 55 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:                                      TTCTGGCTACATCTTCACAACTTATGATATAGACTGGGTGAGGCAGACGCCTGAA55                     (2) INFORMATION FOR SEQ ID NO:29:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 91 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:                                      ACAGGGACTTGAGTGGATTGGATGGATTTTTCCTGGAGAGGGTGGTACTGAATACAATGA60                GAAGTTCAAGGGCAGGGCCACACTGAGTGTA91                                             (2) INFORMATION FOR SEQ ID NO:30:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 71 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:                                      TGTCTATTTCTGTGCTAGAGGGGACTACTATAGGCGCTACTTTGACTTGTGGGGCCAAGG60                GACCACGGTCA71                                                                 (2) INFORMATION FOR SEQ ID NO:31:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 72 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:                                      AGAGGGTCACCATGACCTGCAGTGCCAGCTCAGGTATACGTTACATATATTGGTACCAAC60                AGAAGCCTGGAT72                                                                (2) INFORMATION FOR SEQ ID NO:32:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 63 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:                                      TCCCCCAGACTCCTGATTTATGACACATCCAACGTGGCTCCTGGAGTCCCTTTTCGCTTC60                AGT63                                                                         (2) INFORMATION FOR SEQ ID NO:33:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 68 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:                                      ATGCTGCCACTTATTACTTGCCAGGAGTGGAGTGGTTATCCGTACACGTTCGGAGGGGGG60                ACCAAGCT68                                                                    (2) INFORMATION FOR SEQ ID NO:34:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 55 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:                                      TTCTGGCTACATCTTCACAGAATTCATCTAGATTTGGGTGAGGCAGACGCCTGAA55                     (2) INFORMATION FOR SEQ ID NO:35:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 91 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:                                      ACAGGGACTTGAGTGGATTGGAATCACAGTCAAGCTTATCCTTTATCTCAGGTCTCGAGT60                TCCAAGTACTTAAAGGGCCACACTGAGTGTA91                                             (2) INFORMATION FOR SEQ ID NO:36:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 70 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:                                      TGTCTATTTCTGTGCTAGATCTTGACTGCAGTCTTATACGAGGATCCATTGGGGCCAAGG60                GACCAGGTCA70                                                                  (2) INFORMATION FOR SEQ ID NO:37:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 72 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:                                      AGAGGGTCACCATGACCTGCGGACGTCTTTAAGCGATCGGGCTGATGGCCTGGTACCAAC60                AGAAGCCTGGAT72                                                                (2) INFORMATION FOR SEQ ID NO:38:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 63 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:                                      TCCCCCAGACTCCTGATTTATTAAGGGAGATCTAAACAGCTGTTGGTCCCTTTTCGCTTC60                AGT63                                                                         (2) INFORMATION FOR SEQ ID NO:39:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 67 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:                                      ATGCTGCCACTTATTACTGCTTCTGCGCGCTTAAAGGATATCTTCATTTCGGAGGGGGGA60                CCAAGCT67                                                                     (2) INFORMATION FOR SEQ ID NO:40:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:                                      AGAATTCATCTAGATTTG18                                                          (2) INFORMATION FOR SEQ ID NO:41:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:41:                                      GCTTATCCTTTATCTCAGGTC21                                                       (2) INFORMATION FOR SEQ ID NO:42:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:42:                                      ACTGCAGTCTTATACGAGGAT21                                                       (2) INFORMATION FOR SEQ ID NO:43:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:43:                                      GACGTCTTTAAGCGATCG18                                                          (2) INFORMATION FOR SEQ ID NO:44:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:44:                                      TAAGGGAGATCTAAACAG18                                                          (2) INFORMATION FOR SEQ ID NO:45:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:45:                                      TCTGCGCGCTTAAAGGAT18                                                          (2) INFORMATION FOR SEQ ID NO:46:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 46 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:46:                                      TTCTATTGACGGCCTGTCAGGCCTCATATATACTTTAGATTGATTT46                              (2) INFORMATION FOR SEQ ID NO:47:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 56 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:47:                                      TTGACGCACTGGCCATGGTGGCCAAAAATAAACAAATAGGGGTTCCGCGCACATTT56                    (2) INFORMATION FOR SEQ ID NO:48:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 45 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:48:                                      AACTGACCACGGCCTGACAGGCCGGTCTGACAGTTACCAATGCTT45                               (2) INFORMATION FOR SEQ ID NO:49:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 44 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:49:                                      AACCTGTCCTGGCCACCATGGCCTAAATACATTCAAATATGTAT44                                (2) INFORMATION FOR SEQ ID NO:50:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:50:                                      TTCTTAGACGTCAGGTGGCACTT23                                                     (2) INFORMATION FOR SEQ ID NO:51:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:51:                                      TTTTAAATCAATCTAAAGTAT21                                                       (2) INFORMATION FOR SEQ ID NO:52:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 42 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:52:                                      TGCTCATCCACGAGTGTGGAGAAGTGGTCCCTGCAACTTTAT42                                  (2) INFORMATION FOR SEQ ID NO:53:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 39 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:53:                                      ACCACTTCTCCACACTCGTGGATGAGCACTTTTAAAGTT39                                     (2) INFORMATION FOR SEQ ID NO:54:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 53 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:54:                                      TGTCACTACCACACTCGTGGACTACCATGGCCTAAATACATTCAAATATGTAT53                       (2) INFORMATION FOR SEQ ID NO:55:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:55:                                      TTAAGGGATTTTGGTCATGAGATT24                                                    (2) INFORMATION FOR SEQ ID NO:56:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 141 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:56:                                      GTCGACCTCGAGCCATGGCTAACTAATTAAGTAATNNNTACTGCAGCGTCGTGACTGGGA60                AAACCCTGGGGTTACCCAACTTAATCGCCTTGCTGCGCATCCACCTTTCGCTAGCTGGCG120               GAATTCCGAAGAANNNGCGCG141                                                      (2) INFORMATION FOR SEQ ID NO:57:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 141 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:57:                                      GTCGACCTGCAGGCATGCAAGCTTAGCACTTGCTGTAGTACTGCAGCGTCGTGACTGGGA60                AAACCCTGGGGTTACCCAACTTAATCGCCTTGCTGCGCATCCACCTTTCGCTAGTTAACT120               AATTAACTAAGATATCGCGCG141                                                      (2) INFORMATION FOR SEQ ID NO:58:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 57 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:58:                                      TCGCCTTGCTGCGCATCCACCTTTCGCTAGCTGGCGGAATTCCGAAGAANNNGCGCG57                   (2) INFORMATION FOR SEQ ID NO:59:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 57 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:59:                                      TCGCCTTGCTGCGCATCCACCTTTCGCTAGTTAACTAATTAACTAAGATATCGCGCG57                   (2) INFORMATION FOR SEQ ID NO:60:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 465 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:60:                                      ATGGTTCCGATCCGTCAGCTGCACTACCGTCTGCGTGACGAACAGCAGAAAAGCCTGGTT60                CTGTCCGACCCGTACGAACTGAAAGCTAGGTGATCTTCTCCATGAGCTTCGTACAAGGTG120               AACCAAGCAACGACAAAATCCCGGTGGCTTTGGGTCTGAAAGGTAAAAACCTGTGACCCT180               GCAACTCGAGAGCGTGGACCCAAAACAGTACCCAAAGAAGAAGATGGAGAAGCGTTTCGT240               CTTCAACAAGATCGAAGTCAACCGAACTGGTACATCAGCACCTCCCAAGCAGAGCACAAG300               CCTGTCTTCCTGGGNNNTAACAACTCCGGTCAGGATATCATCGACTTCCTGCACCTGAAT360               GGCCAGAACATCAACCAACACCTGTCCTGTGTAATGAAAGACGGCACTCCGAGCAAAGTG420               GAGTTCGAGTCTGCTGAGTTCACTATGGAATCTGTGTCTTCCTAA465                              (2) INFORMATION FOR SEQ ID NO:61:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 465 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:61:                                      ATGGCACCGGTTAGATCTCTGAACTGCACCCTTCGCGACTCCCAACAGAAAAGCTTAGTA60                ATGTCTGGTCCGTACGAGCTCAAAGCTAGGTTGTATTCAGCATGAGCTTCGTCCAAGGTG120               AAGAGTCTAACGACAAGATCCCAGTTGCATTAGGCCTGAAAGAGAAGAATCTGTGACTCT180               GCAGCTTGAATCCGTTGACCCGAAAAACTATCCGAAGAAGAAAATGGAGAAGCGTTTCGT240               ATTTAACAAGATTGAGATTAACCAAACTGGTACATCAGTACTTCTCAAGCAGAGAATATG300               CCTGTGTTCCTCGGCGGTACCAAAGGCGGTCAGGATATCACTGACTTCCTGCATCTGCAA360               GGCCAGCACATGGAACAACACCTCAGCTGCGTACTGAAAGACGATAAGCCTAACAAGCTG420               GAATTCGAGTCTGCTCAGTTCACCATGCAGTTTGTCTCGAGCTAA465                              (2) INFORMATION FOR SEQ ID NO:62:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: Not Relevant                                                (D) TOPOLOGY: Not Relevant                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:62:                                      GlyGlyGlyGlySer                                                               15                                                                            __________________________________________________________________________

What is claimed is:
 1. A method for forming a mutagenizeddouble-stranded polynucleotide from a template double-strandedpolynucleotide, comprising:a) providing a population of double-strandedoverlapping fragments of the template double-stranded polynucleotide andone or more single or double-stranded oligonucleotides, wherein saidoligonucleotides comprise an area of identity and an area of heterologyto the template double-stranded polynucleotide; b) denaturing theresultant mixture of double-stranded overlapping fragments andoligonucleotides into single-stranded fragments; c) incubating theresultant population of single-stranded fragments with a polymeraseunder conditions which result in the annealing of said single-strandedfragments at said areas of identity to form pairs of annealed fragments,said areas of identity being sufficient for one member of a pair toprime replication of the other thereby forming mutagenizeddouble-stranded polynucleotides; and d) repeating steps (b) and (c) forat least two further cycles, wherein the resultant mixture in step (b)of a further cycle includes the mutagenized double-strandedpolynucleotides from step (c) of the previous cycle, and the furthercycle forms further mutagenized double-stranded polynucleotides wherebythe average length of the mutagenized polynucleotides increases in eachcycle.
 2. The method of claim 1 wherein the concentration of a singlespecies of double-stranded overlapping fragment in the population ofdouble-stranded overlapping fragments is less than 1% by weight of thetotal DNA.
 3. The method of claim 1 wherein the number of differentspecific overlapping double-stranded fragments comprises at least about100.
 4. The method of claim 1 wherein the size of the double-strandedoverlapping fragments is from about 5 bp to 5 kb.
 5. The method of claim1 wherein the size of the template double-stranded polynucleotidecomprises from 50 bp to 100 kb.
 6. The method of claim 1, wherein thedouble-stranded template polynucleotide encodes a wild-type protein; andthe method further comprisesf) expressing a mutant form of the wildtypeprotein from a further mutagenized double-stranded polynucleotide. 7.The method of claim 6 further comprising selecting the desiredrecombinant protein from a population of recombinant proteins encoded byfurther mutagenized polynucleotides produced by repeating cycles of (b)and (c).
 8. A method for obtaining a chimeric polynucleotide sequencecomprising:a) treating a sample comprising different double-strandedtemplate polynucleotides wherein said different template polynucleotidescontain areas of identity and areas of heterology under conditionswhereby overlapping double-stranded fragments of a desired size of saiddifferent double-stranded template polynucleotides are formed; b)denaturing the resultant overlapping double-stranded fragments of saiddifferent double-stranded template polynucleotides contained in thetreated sample produced by step (a) into single-stranded fragments; c)incubating the resultant single-stranded fragments with polymerase underconditions which provide for the annealing of the single-strandedfragments at the areas of identity to form pairs of annealed fragments,said areas of identity being sufficient for one member of a pair toprimer replication of the other thereby forming chimeric double-strandedpolynucleotide sequences comprising template polynucleotide sequences;and d) repeating steps (b) and (c) for at least two cycles, wherein theresultant mixture in step (b) of a cycle includes the chimericdouble-stranded polynucleotide sequences in step (c), and the furthercycle forms a further chimeric polynucleotide sequences whereby and theaverage length of chimeric polynucleotide sequences increases in eachcycle.
 9. The method of claim 8 wherein the concentration of a specificdouble-stranded overlapping fragment in the double-stranded overlappingfragments of a desired size in step (a) is less than 1% by weight of thetotal DNA.
 10. The method of claim 8 where the number of differentdouble-stranded overlapping fragments of a desired size in step (a)comprises at least about
 100. 11. The method of claim 8 wherein the sizeof the double-stranded overlapping fragments of a desired size is fromabout 5 bp to 5 kb.
 12. The method of claim 8 wherein the size of thedifferent template double-stranded polynucleotides is from 50 bp to 100kb.
 13. A method for generating libraries of displayed peptides ordisplayed antibodies suitable for affinity interaction screening orphenotypic screening, the method comprising:(1) obtaining a firstplurality of selected library members comprising a displayed peptide ordisplayed antibody and an associated polynucleotide encoding saiddisplayed peptide or displayed antibody, and obtaining said associatedpolynucleotides or copies thereof wherein said associatedpolynucleotides comprise a region of substantially identical sequence,and (2) pooling and fragmenting said associated polynucleotides orcopies to form a population of double-stranded fragments thereof 3)denaturing the population of double-stranded fragments intosingle-stranded fragments; c) incubating the resultant population ofsingle-stranded fragments with a polymerase under conditions whichresult in the annealing of said single-stranded fragments at areas ofoverlap to form pairs of annealed fragments, whereby one member of apair primes replication of the other thereby forming recombinantpolynucleotides; d) repeating steps b) and c) for at least two furthercycles wherein the resultant mixture in step (b) of a further cycleincludes the recombinant polynucleotides from step c) of the previouscycle, whereby the average length of polynucleotides increases in eachcycle to form a shuffled pool of recombined polynucleotides, whereby asubstantial fraction of the recombined polynucleotides of said shuffledpool are not present in the associated polynucleotides in step (1) andthe shuffled pool of recombined polynucleotides encodes the libraries ofdisplayed peptides or displayed antibodies suitable for affinityinteraction screening or phenotypic screening.
 14. The method of claim13, further comprising introducing mutations into said associatedpolynucleotides or copies.
 15. The method of claim 14, wherein themutations are introduced by performing PCR amplification.
 16. The methodof claim 15, wherein the PCR amplification is error-prone PCR.
 17. Themethod of claim 13, comprising the additional step of screening thelibrary members of the shuffled pool to identify individual shuffledlibrary members having the ability to bind with a predeterminedmacromolecule.
 18. The method of claim 13, wherein the first pluralityof selected library members is obtained by selecting for a phenotypiccharacteristic other than binding affinity for a predetermined molecule.19. The method of claim 13, wherein the first plurality of selectedlibrary members is pooled and fragmented and homologously recombined byPCR in vitro.
 20. The method of claim 13, wherein the first plurality ofselected library members is pooled and fragmented in vitro, theresultant fragments transferred into a host cell or organism andhomologously recombined to form shuffled library members in vivo. 21.The method of claim 13, wherein the first plurality of selected librarymembers is cloned or amplified on episomally replicable vectors, amultiplicity of said vectors is transferred into a cell and homologouslyrecombined to form shuffled library members in vivo.
 22. A method forgenerating libraries of displayed peptides or displayed antibodiessuitable for affinity interaction screening or phenotypic screening, themethod comprising:(1) obtaining a first plurality of selected librarymembers comprising a displayed peptide or displayed antibody and anassociated polynucleotide encoding said displayed peptide or displayedantibody, and obtaining said associated polynucleotides or copiesthereof wherein said associated polynucleotides comprise a region ofsubstantially identical sequence, and (2) cloning or amplifying saidassociated polynucleotides or copies on episomally replicable vectorsand transferring a multiplicity of said vectors into a cell andhomologously recombining to form shuffled library members in vivoencoding the libraries of displayed peptide or displayed antibodiessuitable for affinity interaction screening or phenotypic screening. 23.The method of claim 22, further comprising introducing mutations intosaid associated polynucleotides or copies thereof.
 24. The method ofclaim 22, wherein said episomally replicable vectors comprise a directrepeat of a plurality of associated polynucleotides or copies thereof.25. The method of claim 13 for generating libraries of displayedantibodies, wherein the shuffled pool of recombined polynucleotidescomprises novel combinations of CDRs, whereby a substantial fraction ofthe recombined polynucleotides of said shuffled pool comprise CDRcombinations are not present in the first plurality of selected librarymembers.
 26. The method of claim 25, comprising the additional stepwherein the shuffled pool is subjected to affinity screening to selectshuffled library members which bind to a predetermined epitope andthereby selecting a plurality of selected shuffled library members. 27.The method of claim 26, comprising the additional step of shuffling theplurality of selected shuffled library members and screening, from 1 toabout 1000 cycles.
 28. The method of claim 1, wherein step d) comprisesrepeating steps b)-c) for at least 10 cycles.